Integral membrane protien display on poxvirus extracellular enveloped virions

ABSTRACT

This disclosure provides compositions and methods for expressing and displaying isolated integral membrane proteins (IMPs) or fragments thereof in a native conformation for use in the screening, selecting, and identifying of antibodies or antibody-like molecules that bind to a target IMP of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/384,087, filed Apr. 15, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/091,077, filed Oct. 3, 2018, which is a U.S.National Stage Entry of PCT Application No. PCT/US2017/028787, filedApr. 21, 2017, which claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/326,501 filed on Apr. 22,2016, which are each hereby incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name “Sequence Listing.txt; Size: 61,440 bytes; and Date ofCreation: Oct. 3, 2018”) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND

Antibodies of defined specificity are being employed in an increasingnumber of diverse therapeutic applications. A number of methods havebeen used to obtain useful antibodies for human therapeutic use. Theseinclude chimeric and humanized antibodies, and fully human antibodiesselected from libraries, e.g., phage display libraries, or fromtransgenic animals. Immunoglobulin libraries constructed inbacteriophage can derive from antibody producing cells of naïve orspecifically immunized individuals and could, in principle, include newand diverse pairings of human immunoglobulin heavy and light chains.Although this strategy does not suffer from an intrinsic repertoirelimitation, it requires that complementarity determining regions (CDRs)of the expressed immunoglobulin fragment be synthesized and foldproperly in bacterial cells. Many antigen binding regions, however, aredifficult to assemble correctly as a fusion protein in bacterial cells.In addition, the protein will not undergo normal eukaryoticpost-translational modifications. As a result, this method imposes adifferent selective filter on the antibody specificities that can beobtained. Alternatively, fully human antibodies can be isolated fromlibraries in eukaryotic systems, e.g., yeast display, retroviraldisplay, or expression in DNA viruses such as poxviruses. See, e.g.,U.S. Pat. No. 7,858,559, and U.S. Patent Appl. Publication No.2013-028892, which are incorporated herein by reference in theirentireties.

Many important targets for therapeutic antibodies are integral membraneproteins (IMPs), e.g., multi-pass membrane proteins (GPCRs, IonChannels, etc.) that are difficult to express and purify in aconformationally-intact state. The absence of properly folded targetproteins in an isolated state makes the identification and selection ofantibodies to these targets challenging. While certain IMPs can beexpressed on the surface of cells, e.g., mammalian cells, whole cellsare problematic for use in antibody discovery because they are complexantigen mixtures, target expression can be low, and because certaindisplay packages used to construct antibody libraries (e.g., vacciniavirus antibody libraries) can bind to whole cells non-specifically.There remains a need for new methods to express and display target IMPsof interest in their native conformation at a sufficient concentrationand with minimal competition from other cell proteins to allow foridentification and selection of therapeutic antibodies and antibody-likemolecules from display libraries.

SUMMARY

This disclosure provides compositions and methods for expressing anddisplaying isolated integral membrane proteins (IMPs) or fragmentsthereof in a native conformation for use in the screening, selecting,and identifying of antibodies or antibody-like molecules that bind to atarget IMP of interest.

In certain embodiments, the disclosure provides an isolatedpolynucleotide that includes: a first nucleic acid fragment that encodesan integral membrane protein (IMP) or fragment thereof, where the IMP orfragment thereof includes at least one extra-membrane region, at leastone transmembrane domain and at least one intra-membrane region, andwhere a portion of the first nucleic acid fragment encoding at least oneintra-membrane region is situated at the 5′ or 3′ end of the firstnucleic acid fragment; and a second nucleic acid fragment that encodes avaccinia virus F13L protein or functional fragment thereof, where thesecond nucleic acid fragment is fused in frame to a portion of the firstnucleic acid fragment that encodes an intra-membrane region of the IMP.According to these embodiments, a poxvirus infected cell containing thepolynucleotide can express an IMP-F13L fusion protein as part of theouter envelope membrane of an extracellular enveloped virion (EEV). Incertain aspects, the F13L protein or functional fragment thereof caninclude the amino acid sequence SEQ ID NO: 1 or a functional fragmentthereof. In certain aspects the IMP is a multi-pass membrane proteincomprising at least two, at least three, at least four, at least five,at least six or at least seven transmembrane domains. In certain aspectsthe IMP is a multi-pass membrane protein listed in Table 1.

In certain aspects the multi-pass IMP can have an odd number oftransmembrane domains, the 5′ end of the first nucleic acid fragment canencode an extra-membrane region, and the 3′ end of the first nucleicacid fragment can encode an intra-membrane region fused to the 5′ end ofthe second nucleic acid fragment. In certain aspects the first nucleicacid fragment of this type can encode, e.g., a G-protein coupledreceptor (GPCR). In certain aspects the GPCR can be the human frizzled-4protein (FZD4), or a fragment thereof, and the polynucleotide can encodea polypeptide that includes amino acids 20 to 892 of SEQ ID NO: 2. Incertain aspects the polypeptide can further include a signal peptide,e.g., amino acids 1 to 19 of SEQ ID NO: 2. In certain aspects the GPCRcan be a CXC chemokine receptor, e.g., CXCR4, or a fragment thereof, andthe polynucleotide can encode a polypeptide that includes the amino acidsequence SEQ ID NO: 3.

In certain aspects the multi-pass IMP can have an even number oftransmembrane domains, and both the 5′ and 3′ ends of the first nucleicacid fragment can encode intra-membrane regions. In certain aspects, thesecond nucleic acid fragment can be fused to 3′ end of the first nucleicacid fragment. In certain aspects the IMP can be, e.g., human CD20protein, or a fragment thereof, and the polynucleotide can encode apolypeptide that includes the amino acid sequence SEQ ID NO: 4.

In certain aspects, the first and second nucleic acid fragments of apolynucleotide provided herein can be directly fused. In certain aspectsthe polynucleotide as provided herein can include a third nucleic acidfragment encoding a heterologous peptide, e.g., a linker sequence, anamino acid tag or label, or a peptide or polypeptide sequence thatfacilitates purification, such as a histidine tag. In certain aspects apolynucleotide as provided here can be operably associated with apoxvirus promoter, e.g., a p7.5, a T7, or H5 promoter.

The disclosure further provides an F13L fusion protein encoded by apolynucleotide as provided herein. The disclosure further provides apoxvirus genome, e.g., a vaccinia virus genome, that includes apolynucleotide as provided herein. The disclosure further provides arecombinant vaccinia virus EEV that includes a poxvirus genome asprovided herein.

The disclosure further provides a method of producing a recombinantvaccinia virus EEV as provided herein where the method includesinfecting a host cell permissive for vaccinia virus infectivity with avaccinia virus comprising a poxvirus genome as provided herein, andrecovering EEV released from the host cell.

The disclosure further provides a method to display an integral membraneprotein (IMP) or fragment thereof in a native conformation where themethod includes infecting host cells permissive for poxvirus infectivitywith a recombinant poxvirus that expresses an IMP or fragment thereof asa fusion protein with poxvirus EEV-specific protein ormembrane-associated fragment thereof, where EEV produced by the infectedhost cell comprise the IMP fusion protein as part of the EEV outerenvelope membrane and recovering EEV released from the host cell. Incertain aspects the IMP or fragment thereof displays on the surface ofthe EEV in a native conformation. In certain aspects the EEV-specificprotein can be the vaccinia virus A33R protein, A34R protein, A56Rprotein, B5R protein, A36R protein, F13L protein, anymembrane-associated fragment thereof, or any combination thereof.

In certain aspects the EEV-specific protein is F13L (SEQ ID NO: 1) or afunctional fragment thereof. In certain aspects the IMP is a multi-passmembrane protein that includes at least two, at least three, at leastfour, at least five, at least six or at least seven transmembranedomains. In certain aspects the IMP can be a G-protein coupled receptor(GPCR), e.g., human FZD4 or CXCR4 as described above, that includesseven transmembrane domains, and the F13L protein can be fused to theC-terminus of the IMP. In certain aspects the IMP or fragment thereofcan have an even number of transmembrane domains, e.g., human CD20 asdescribed above, where both the N-terminus and the C-terminus of the IMPor fragment thereof are intra-membrane, and the F13L can be fused to theN-terminus or the C-terminus of the IMP.

In certain aspects the membrane-associated EEV specific protein fragmentcan include or consist of the stalk, transmembrane, and intra-membranedomains of the vaccinia virus A56R protein, e.g., amino acids 108 to 314of SEQ ID NO: 5. In certain aspects IMP portion of the A56R fusionprotein can include the extracellular domain of human FZD4, e.g., thefusion protein can include amino acids 20 to 370 of SEQ ID NO: 6, theextracellular domain of human ErbB2 (Her2), e.g., the fusion protein caninclude amino acids 20 to 855 of SEQ ID NO: 7, or the extracellulardomain of human CD100 (Semaphorin 4D), e.g., the fusion protein caninclude amino acids 20 to 935 of SEQ ID NO: 8.

In certain aspects the membrane-associated EEV specific protein fragmentcan include or consist of the transmembrane and intra-membrane domains,or the stalk, transmembrane, and intra-membrane domains of the vacciniavirus B5R protein, e.g., amino acids 276 to 317 of SEQ ID NO: 9 or aminoacids 238 to 317 of SEQ ID NO: 9, respectively. In certain aspects theIMP portion of the B5R fusion protein can include the extracellulardomain of human FZD4, e.g., the fusion protein can include amino acids20 to 243 of SEQ ID NO: 10 or amino acids 20 to 281 of SEQ ID NO: 11.

The disclosure further provides a fusion protein comprising: amino acids20 to 892 of SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; amino acids 20 to370 of SEQ ID NO: 6; amino acids 20 to 855 of SEQ ID NO: 7; amino acids20 to 935 of SEQ ID NO: 8; amino acids 20 to 243 of SEQ ID NO: 10; aminoacids 20 to 281 of SEQ ID NO: 11, amino acids 20 to 506 of SEQ ID NO:16, or amino acids 20 to 235 of SEQ ID NO: 17. A fusion protein asprovided, when expressed by a recombinant poxvirus, e.g., a vacciniavirus, can appear on the surface of a poxvirus extracellular envelopedvirion (EEV) in a native conformation. A recombinant poxvirus EEVcomprising the fusion protein is also provided. The disclosure furtherprovides a recombinant poxvirus EEV that includes a heterologous IMP orfragment thereof fused to a poxvirus EEV-specific protein ormembrane-associated fragment thereof, where the fusion protein issituated in the EEV outer envelope membrane, and where the IMP orfragment thereof displays on the surface of the EEV in its nativeconformation. In certain aspects the recombinant poxvirus EEV is avaccinia virus EEV.

The disclosure further provides a method to select antibodies that bindto a multi-pass membrane protein where the method includes attachingrecombinant EEV as provided herein to a solid support; providing anantibody display library, where the library comprises display packagesdisplaying a plurality of antigen binding domains; contacting thedisplay library with the EEV such that display packages displayingantigen binding domains that specifically binds to the IMP expressed onthe EEV can bind thereto; removing unbound display packages; andrecovering display packages that display an antigen binding domainspecific for the IMP expressed on the EEV. In certain aspects of thismethod the recombinant EEV are inactivated prior to attachment to thesolid support, e.g., by incubation with Psoralen (Trioxsalen,4′-aminomethyl-, hydrochloride) in the presence of UV irradiation. Incertain aspects of this method the recombinant EEV are attached to thesolid surface via reaction with tosyl groups attached to the surface. Incertain aspects the solid surface can be tosyl-activated magnetic beads.In certain aspects of this method the recombinant EEV are biotinylatedand attached to a streptavidin coated solid surface, e.g.,streptavidin-coated magnetic beads.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A-C: Diagrammatic depiction of integral membrane proteins (IMPs)or fragment thereof fused to vaccinia virus extracellular envelopedvirion (EEV)-specific proteins or fragments thereof The parallelhorizontal lines are a diagram of the EEV outer membrane. FIG. 1Adiagrams the extracellular domain (ECD) of an IMP fused to a fragment ofthe vaccinia A56R protein that includes the transmembrane domain and theintra-membrane domain. FIG. 1B diagrams the topology of a typical Gprotein-coupled receptor fused to the vaccinia virus EEV-specific F13Lprotein. F13L is associated with the inner side of the EEV outermembrane via palmitoylation. FIG. 1C diagrams the topology of an IMPwith an even number of transmembrane domains, e.g., CD20), fused toF13L.

FIG. 2: Demonstration of incorporation of CD20-F13L and CD20 ECD-A56Rfusion proteins into vaccinia virus EEV particles.

FIG. 3A: Demonstration of preferential incorporation of CD20-F13L fusionprotein over untagged CD20 into vaccinia virus EEV particles.

FIG. 3B: Demonstration of preferential incorporation of FZD4-F13L fusionprotein over untagged (unfused) FZD4 into vaccinia virus EEV particles.

FIG. 4: Incorporation of additional IMP-EEV protein fusions intovaccinia virus EEV. “CD20” is a CD20-F13L fusion protein, “CXCR4” is aCXCR4-F13L fusion protein, “Her2” is a Her2 ECD-A56R fusion protein; and“CD100” is a CD100 ECD-A56R fusion protein.

FIG. 5: Outline of assay for screening an antibody display library fordisplay packages that bind to an IMP of interest expressed on vacciniavirus EEV.

FIG. 6A: Binding of vaccinia virus EEV expressing an anti-HER-2 antibodyto vaccinia virus EEV expressing the HER2 ECD as a fusion with thevaccinia virus A56R protein, bound by tosyl-groups to magnetic beads.

FIG. 6B: Binding of vaccinia virus EEV expressing an anti-FZD antibodyto vaccinia virus EEV expressing FZD4 as a fusion with the vacciniavirus F13L protein, bound by tosyl-groups to magnetic beads.

FIG. 6C: Binding of vaccinia virus EEV expressing an anti-CXCR4 antibodyto vaccinia virus EEV expressing the CXCR4 as a fusion with the vacciniavirus F13L protein, bound by tosyl-groups to magnetic beads.

FIG. 6D: Binding of vaccinia virus EEV expressing an anti-CD100 (“sema”)antibody to vaccinia virus EEV expressing the CD100 ECD as a fusion withthe vaccinia virus A56R protein, bound by tosyl-groups to magneticbeads.

FIG. 7: FACS scans showing enrichment for anti FZD4 antibodies followingpanning on inactivated FZD-ECD-A45R-expressing EEV bound by tosyl-groupsto magnetic beads after 3 (Rd3), 4 (Rd4), and 5 (Rd5) rounds of panning.The top row shows antibody-expressing virus-infected cells stained with10 μg/ml FZD-His, followed by anti-His-Dyelight650 and anti-Fab-FITC.The bottom row shows antibody-expressing virus-infected cells stainedwith 10 μg/ml CD100-His (negative control), followed byanti-His-Dyelight650 and anti-Fab-FITC.

FIG. 8: Incorporation two different protein fusions (HA-A56R fusion andFZD4-F13L fusion) into vaccinia virus EEV. EEV expressing the HA-A56Rfusion alone, the FZD4-F13L fusion alone, or both fusion proteins, weretested for binding to either anti-FZD4-coated beads or anti-HA coatedbeads.

FIG. 9: Specific recovery of anti-CXCR4-expressing EEV by magnetic beadscoated with EEV expressing both an HA-A56R fusion and CXCR4-F13L fusion.The antigen-EEV were coupled to anti-HA coated beads.

FIG. 10: Binding of biotinylated vaccinia virus EEV expressing thedesignated fusion proteins to streptavidin coated magnetic beads.

DETAILED DESCRIPTION

This disclosure provides methods and compositions for expressing anddisplaying integral membrane proteins (IMPs), e.g., multi-pass (IMPs),in a conformationally intact or native state on the surface ofextracellular enveloped virion particles (EEV) of poxviruses, e.g.,vaccinia virus, as a fusion with a polypeptide segment an EEV-specificmembrane-associated protein, e.g., F13L.

Definitions

The term “a” or “an” entity refers to one or more of that entity; forexample, “a binding molecule,” is understood to represent one or morebinding molecules. As such, the terms “a” (or “an”), “one or more,” and“at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

As used herein, the term “non-naturally occurring” substance,composition, entity, and/or any combination of substances, compositions,or entities, or any grammatical variants thereof, is a conditional termthat explicitly excludes, but only excludes, those forms of thesubstance, composition, entity, and/or any combination of substances,compositions, or entities that are well-understood by persons ofordinary skill in the art as being “naturally-occurring,” or that are,or might be at any time, determined or interpreted by a judge or anadministrative or judicial body to be, “naturally-occurring.”

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation, andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a biological source or produced byrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide as disclosed herein can be of a size of about 3 or more, 5or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid, e.g., a serine or an asparagine.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated as disclosed herein, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, the term “non-naturally occurring” polypeptide, or anygrammatical variants thereof, is a conditional term that explicitlyexcludes, but only excludes, those forms of the polypeptide that arewell-understood by persons of ordinary skill in the art as being“naturally-occurring,” or that are, or might be at any time, determinedor interpreted by a judge or an administrative or judicial body to be,“naturally-occurring.”

Other polypeptides disclosed herein are fragments, derivatives, analogs,or variants of the foregoing polypeptides, and any combination thereofThe terms “fragment,” “variant,” “derivative” and “analog” as disclosedherein include any polypeptides that retain at least some of theproperties of the corresponding native antibody or polypeptide, forexample, specifically binding to an antigen. Fragments of polypeptidesinclude, for example, proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of, e.g., a polypeptide include fragments asdescribed above, and also polypeptides with altered amino acid sequencesdue to amino acid substitutions, deletions, or insertions. In certainaspects, variants can be non-naturally occurring. Non-naturallyoccurring variants can be produced using art-known mutagenesistechniques. Variant polypeptides can comprise conservative ornon-conservative amino acid substitutions, deletions or additions.Derivatives are polypeptides that have been altered so as to exhibitadditional features not found on the original polypeptide. Examplesinclude fusion proteins. Variant polypeptides can also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of apolypeptide can also refer to a subject polypeptide having one or moreamino acids chemically derivatized by reaction of a functional sidegroup. Also included as “derivatives” are those peptides that containone or more derivatives of the twenty standard amino acids. For example,4-hydroxyproline can be substituted for proline; 5-hydroxylysine can besubstituted for lysine; 3-methylhistidine can be substituted forhistidine; homoserine can be substituted for serine; and ornithine canbe substituted for lysine.

A “conservative amino acid substitution” is one in which one amino acidis replaced with another amino acid having a similar side chain.Families of amino acids having similar side chains have been defined inthe art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the present disclosure do not abrogatethe binding of the polypeptide or antibody containing the amino acidsequence, to the antigen to which the binding molecule binds. Methods ofidentifying nucleotide and amino acid conservative substitutions that donot eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32:1180-1 187 (1993); Kobayashi et al.,Protein Eng. 12 (10):879-884 (1999); and Burks et al., Proc. Natl. Acad.Sci. USA 94:412-417 (1997)).

As used herein the term “integral membrane protein” or “IMP” refers to aprotein or polypeptide that is attached to a biological membrane. Oneexample of an IMP is a transmembrane protein, which spans the lipidbilayer of the biological membrane one or more times. Single-passmembrane proteins cross the membrane only once, while multi-passmembrane proteins weave in and out, crossing several times. Type Isingle-pass proteins are positioned with their amino terminus on theouter side of the membrane or “extra-membrane” and theircarboxyl-terminus on the interior side of the membrane, or“intra-membrane.” Type II single-pass proteins have their amino-terminuson the intra-membrane side. Multi-pass transmembrane proteins passthrough the membrane two or more times and can have a variety ofdifferent topologies. Those proteins with an even number oftransmembrane domains will have both their amino terminus and carboxyterminus on the same side of the membrane. One example of such a proteinis CD20, which is expressed on B cells. Those with an odd number oftransmembrane domains will have their amino-and carboxy termini onopposite sides of the membrane. Examples include G-protein coupledreceptors, which typically have 7 transmembrane domains, with the aminoterminus on the extra-membrane side and the carboxy terminus on theintra-membrane side. Certain IMPs do not have transmembrane domains andare instead anchored to the membrane, e.g., via a lipid such asglycosylphosphatidylinositol or palmitoyl group. IMPs have myriadbiological functions including, but not limited to transporters,linkers, channels, receptors, enzymes, energy transduction or celladhesion.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmidDNA (pDNA). A polynucleotide can comprise a conventional phosphodiesterbond or a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acidsequence” refer to any one or more nucleic acid segments, e.g., DNA orRNA fragments, present in a polynucleotide.

By an “isolated” nucleic acid or polynucleotide is intended any form ofthe nucleic acid or polynucleotide that is separated from its nativeenvironment. For example, gel-purified polynucleotide, or a recombinantpolynucleotide encoding a polypeptide contained in a vector would beconsidered to be “isolated.” Also, a polynucleotide segment, e.g., a PCRproduct, that has been engineered to have restriction sites for cloningis considered to be “isolated.” Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in a non-native solution such as a buffer or saline.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofpolynucleotides, where the transcript is not one that would be found innature. Isolated polynucleotides or nucleic acids further include suchmolecules produced synthetically. In addition, polynucleotide or anucleic acid can be or can include a regulatory element such as apromoter, ribosome binding site, or a transcription terminator.

As used herein, a “non-naturally occurring” polynucleotide, or anygrammatical variants thereof, is a conditional definition thatexplicitly excludes, but only excludes, those forms of thepolynucleotide that are well-understood by persons of ordinary skill inthe art as being “naturally-occurring,” or that are, or that might be atany time, determined or interpreted by a judge or an administrative orjudicial body to be, “naturally-occurring.”

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g., on a single vector, or in separate polynucleotideconstructs, e.g., on separate (different) vectors. Furthermore, anyvector can contain a single coding region, or can comprise two or morecoding regions, e.g., a single vector can separately encode animmunoglobulin heavy chain variable region and an immunoglobulin lightchain variable region. In addition, a vector, polynucleotide, or nucleicacid can include heterologous coding regions, either fused or unfused toanother coding region. Heterologous coding regions include withoutlimitation, those encoding specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally can include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter can be a cell-specific promoter thatdirects substantial transcription of the DNA in predetermined cells.Other transcription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions that function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Poxvirus promoters (e.g. p7.5 or H5) or the bacteriophage T7 promotercan also be used as transcription control regions. When employing a T7promoter, an inducible vaccinia expression system can be utilized. Thevaccinia expression system can include, but is not limited, to a firstrecombinant vaccinia virus that encodes the entire bacteriophage T7 gene1 coding region for T7 RNA polymerase, and a second recombinant vacciniavirus that encodes a gene of interest flanked by a T7 promoter andtermination regulatory elements. Dual infection of eukaryotic cells withboth recombinant vaccinia viruses results in synthesis of the T7 RNApolymerase and expression of the gene of interest controlled by the T7promoter.

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide can be RNA, for example, in theform of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

Polynucleotide and nucleic acid coding regions can be associated withadditional coding regions that encode secretory or signal peptides,which direct the secretion of a polypeptide encoded by a polynucleotideas disclosed herein. According to the signal hypothesis, proteinssecreted by mammalian cells have a signal peptide or secretory leadersequence that is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells can have a signal peptidefused to the N-terminus of the polypeptide, which is cleaved from thecomplete or “full length” polypeptide to produce a secreted or “mature”form of the polypeptide. In certain embodiments, the native signalpeptide, e.g., an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, can be used. Forexample, the wild-type leader sequence can be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

As used herein, a “library” is a representative genus ofpolynucleotides, e.g., a group of polynucleotides related through, forexample, their origin from a single animal species, tissue type, organ,or cell type, where the library collectively comprises at least twodifferent species within a given genus of polynucleotides. A library ofpolynucleotides can include, e.g., at least two, at least 5, at least10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different species within agiven genus of polynucleotides. In certain aspects, a library ofpolynucleotides as provided herein can encode a plurality ofpolypeptides that contains a polypeptide of interest. In certainaspects, a library of polynucleotides as provided herein can encode aplurality of immunoglobulin subunit polypeptides, e.g., heavy chainsubunit polypeptides or light chain subunit polypeptides. In thiscontext, a “library” as provided herein comprises polynucleotides of acommon genus, the genus being polynucleotides encoding immunoglobulinsubunit polypeptides of a certain type and class e.g., a library mightencode a human μ, γ-1, γ-2, γ-3, γ-4, α-1, α-2, ε, or δ heavy chain, ora human κ or λ light chain. Although each member of any one libraryconstructed according to the methods provided herein can encode the sameheavy or light chain constant region and/or a membrane anchoring domain,the library can collectively comprise at least two, at least 5, or atleast 10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different variableregion associated with the common constant region.

In other embodiments, the library can a plurality of immunoglobulinsingle-chain fragments that comprise a variable region, such as a lightchain variable region or a heavy chain variable region, and/or both alight chain variable region and a heavy chain variable region, e.g., anScFv fragment.

As used herein, a “display library” is a library of polynucleotides eachcarried in a “display package” that expresses the polypeptide encoded bythe library polynucleotide on its surface. An antibody display library,for example, can include plurality of display packages, each displayingan antigen binding domain of an antibody on its surface. When thedisplay library is permitted to interact with an antigen of interest,e.g., immobilized on a solid surface, those display packages that bindthe antigen can be isolated from the rest of the library and recovered.The polynucleotide encoding the antigen binding domain displayed on thesurface of the display package can then be isolated. Display librariesinclude, without limitation, phage display libraries in bacteria orlibraries in eukaryotic systems, e.g., yeast display, retroviraldisplay, or expression in DNA viruses such as poxviruses. See, e.g.,U.S. Pat. No. 7,858,559, and U.S. Patent Appl. Publication No.2013-028892, which are incorporated herein by reference in theirentireties. In certain aspects, an antibody display library can beprepared in a poxvirus, e.g., vaccinia virus vector, as fusion proteinswith an EEV-specific protein, such that the “display packages” are EEVparticles. See U.S. Patent Appl. Publication No. 2013-028892.

Such display libraries can be screened against the IMP fusion proteinsdisplayed on the surface of EEV as provided herein.

By “recipient cell” or “host cell” or “cell” is meant a cell orpopulation of cells in which a recombinant protein can be expressed, avirus can be propagated, or polynucleotide libraries as provided hereincan be constructed and/or propagated. A host cell as provided herein istypically a eukaryotic cell or cell line, e.g., a vertebrate, mammalian,rodent, mouse, primate, or human cell or cell line. By “a population ofhost cells” is meant a group of cultured cells which a “library” asprovided herein can be constructed, propagated, and/or expressed. Anyhost cell which is permissive for vaccinia virus infectivity is suitablefor the methods provided by this disclosure. Host cells for use in themethods provided herein can be adherent, e.g., host cells that growattached to a solid substrate, or, alternatively, the host cells can bein suspension.

Host cells as provided herein can comprise a constitutive secretorypathway, where proteins, e.g., proteins of interest expressed by thecell or by a library, are secreted from the interior of the cell eitherto be expressed on a cell or viral membrane surface or to be fullysecreted as soluble polypeptides. In certain aspects, proteins ofinterest expressed on or in a biological membrane, e.g., an IMP, areexpressed on the surface of an enveloped virus produced by the hostcell, e.g., an extracellular enveloped vaccinia virus, or EEV. IMPS canfollow the same pathway as fully secreted forms or proteins, passingthrough to the ER lumen, except that they can be retained in the ERmembrane by the presence of one or more stop-transfer signals, or“transmembrane domains.” Transmembrane domains are hydrophobic stretchesof about 20 amino acids that adopt an alpha-helical conformation as theytransverse the membrane. Membrane embedded proteins are anchored in thephospholipid bilayer of the plasma membrane. Transmembrane forms ofpolypeptides of interest, e.g., membrane-anchored immunoglobulin heavychain polypeptides typically utilize amino terminal signal peptides asdo fully secreted forms.

Signal peptides, transmembrane domains, and cytosolic or“intra-membrane” domains are known for a wide variety of membrane boundand/or fully secreted proteins.

Suitable transmembrane domains can include, but are not limited to theTM domain of the vaccinia virus EEV-specific HA protein A56R, or theEEV-specific vaccinia virus transmembrane proteins A33R, A34R, A36R, orB5R. See, e.g., U.S. Patent Appl. Publ. No. 2013/0288927, published Oct.31, 2013, and incorporated herein by reference in its entirety. Incertain aspects the EEV specific protein can be anchored to the innersurface of the viral envelope via a palmitoyl group, e.g., the vacciniavirus protein F13L, discussed in more detail elsewhere herein.

As used herein, the term “binding molecule” refers in its broadest senseto a molecule that specifically binds to a receptor, e.g., an epitope oran antigenic determinant. As described further herein, a bindingmolecule can comprise one or more “antigen binding domains” describedherein. A non-limiting example of a binding molecule is an antibody orfragment thereof that retains antigen-specific binding.

The terms “binding domain” and “antigen binding domain” are usedinterchangeably herein and refer to a region of a binding molecule thatis necessary and sufficient to specifically bind to an epitope. Forexample, an “Fv,” e.g., a variable heavy chain and variable light chainof an antibody, either as two separate polypeptide subunits or as asingle chain, is considered to be a “binding domain.”

Other antigen binding domains include, without limitation, the variableheavy chain (VHH) of an antibody derived from a camelid species, or siximmunoglobulin complementarity determining regions (CDRs) expressed in afibronectin scaffold.

The terms “antibody” and “immunoglobulin” can be used interchangeablyherein. An antibody (or a fragment, variant, or derivative thereof asdisclosed herein) includes at least the variable region of a heavy chain(e.g., for camelid species) or at least the variable regions of a heavychain and a light chain. Basic immunoglobulin structures in vertebratesystems are relatively well understood. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988). Unless otherwise stated, the term “antibody” encompassesanything ranging from a small antigen binding fragment of an antibody toa full sized antibody, e.g., an IgG antibody that includes two completeheavy chains and two complete light chains.

The term “immunoglobulin” comprises various broad classes ofpolypeptides that can be distinguished biochemically. Those skilled inthe art will appreciate that heavy chains are classified as gamma, mu,alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some subclasses amongthem (e.g., γ1-γ4 or α1-α2)). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, IgA₂, etc. are well characterized and are known toconfer functional specialization.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. The basicstructure of certain antibodies, e.g., IgG antibodies, includes twoheavy chain subunits and two light chain subunits covalently connectedvia disulfide bonds to form a “Y” structure, also referred to herein asan “H2L2” structure.

The term “epitope” includes any molecular determinant capable ofspecific binding to an antibody. In certain aspects, an epitope caninclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl, or sulfonyl, and, in certainaspects, can have three dimensional structural characteristics, and orspecific charge characteristics. An epitope is a region of a target thatis bound by an antibody.

The term “target” is used in the broadest sense to include substancesthat can be bound by a binding molecule. A target can be, e.g., apolypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule.Moreover, a “target” can, for example, be a cell, an organ, or anorganism that comprises an epitope bound that can be bound by a bindingmolecule.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variableregions (which can be called “variable domains” interchangeably herein)of both the variable light (VL) and variable heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (e.g., CH1, CH2 orCH3) confer biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 (or CH4 in the case ofIgM) and CL domains are at the carboxy-terminus of the heavy and lightchain, respectively.

The six “complementarity determining regions” or “CDRs” present in anantibody antigen binding domain are short, non-contiguous sequences ofamino acids that are specifically positioned to form the antigen bindingdomain as the antibody assumes its three dimensional configuration in anaqueous environment. The remainder of the amino acids in the antigenbinding domain, referred to as “framework” regions, show lessinter-molecular variability. The framework regions largely adopt aβ-sheet conformation and the CDRs form loops that connect, and in somecases form part of, the β-sheet structure. Thus, framework regions actto form a scaffold that provides for positioning the CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto its cognate epitope. The amino acids that make up the CDRs and theframework regions, respectively, can be readily identified for any givenheavy or light chain variable region by one of ordinary skill in theart, since they have been defined in various different ways (see,“Sequences of Proteins of Immunological Interest,” Kabat, E., et al.,U.S. Department of Health and Human Services, (1983); and Chothia andLesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated hereinby reference in their entireties).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. These particular regionshave been described, for example, by Kabat et al., U.S. Dept. of Healthand Human Services, “Sequences of Proteins of Immunological Interest”(1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), whichare incorporated herein by reference. Immunoglobulin variable domainscan also be analyzed, e.g., using the IMGT information system(www://imgt.cines.fr/) (IMGT®/V-Quest) to identify variable regionsegments, including CDRs. (See, e.g., Brochet et al., Nucl. Acids Res.,36:W503-508, 2008).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless use of the Kabat numbering system is explicitly noted, however,consecutive numbering is used for all amino acid sequences in thisdisclosure.

Binding molecules, e.g., antibodies or antigen binding fragments,variants, or derivatives thereof include, but are not limited to,polyclonal, monoclonal, human, humanized, or chimeric antibodies, singlechain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), single domain antibodies such as camelidVHH antibodies, fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library. ScFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulinor antibody molecules encompassed by this disclosure can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Alsocontemplated are immunoglobulin new antigen receptor (IgNAR) isotypesthat are bivalent and comprise a single chain that includes an IgNARvariable domain (VNAR). (See, Walsh et al., Virology 411:132-141, 2011).

By “specifically binds,” it is generally meant that a binding molecule,e.g., an antibody or fragment, variant, or derivative thereof binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, a binding molecule is said to“specifically bind” to an epitope when it binds to that epitope, via itsantigen binding domain more readily than it would bind to a random,unrelated epitope. The term “specificity” is used herein to qualify therelative affinity by which a certain binding molecule binds to a certainepitope. For example, binding molecule “A” can be deemed to have ahigher specificity for a given epitope than binding molecule “B,” orbinding molecule “A” can be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with one or more antigen bindingdomains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population of antigenbinding domains and an antigen. See, e.g., Harlow at pages 29-34.Avidity is related to both the affinity of individual antigen bindingdomains in the population with specific epitopes, and also the valenciesof the immunoglobulins and the antigen. For example, the interactionbetween a bivalent monoclonal antibody and an antigen with a highlyrepeating epitope structure, such as a polymer, would be one of highavidity. An interaction between a between a bivalent monoclonal antibodywith a receptor present at a high density on a cell surface would alsobe of high avidity.

As used herein, the term “heavy chain subunit” or “heavy chain domain”includes amino acid sequences derived from an immunoglobulin heavychain, a binding molecule, e.g., an antibody comprising a heavy chainsubunit can include at least one of: a VH domain, a CH1 domain, a hinge(e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, aCH3 domain, a CH4 domain, or a variant or fragment thereof.

As used herein, the term “light chain subunit” or “light chain domain”includes amino acid sequences derived from an immunoglobulin lightchain. The light chain subunit includes at least one of a VL or CL(e.g., Cκ or Cλ) domain.

Binding molecules, e.g., antibodies or antigen binding fragments,variants, or derivatives thereof can be described or specified in termsof the epitope(s) or portion(s) of an antigen that they recognize orspecifically bind. The portion of a target antigen that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target antigen can comprisea single epitope or at least two epitopes, and can include any number ofepitopes, depending on the size, conformation, and type of antigen.

As used herein, the terms “linked,” “fused” or “fusion” or othergrammatical equivalents can be used interchangeably. These terms referto the joining together of two more elements or components, by whatevermeans including chemical conjugation or recombinant means. An “in-framefusion” refers to the joining of two or more polynucleotide open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the translational reading frame of the original ORFs. Thus, arecombinant fusion protein is a single protein containing two or moresegments that correspond to polypeptides encoded by the original ORFs(which segments are not normally so joined in nature). Although thereading frame is thus made continuous throughout the fused segments, thesegments can be physically or spatially separated by, for example,in-frame linker sequence. For example, polynucleotides encoding an IMPand a vaccinia virus EEV-specific protein can be fused, in-frame, but beseparated by a polynucleotide encoding a linker or spacer, as long asthe “fused” open reading frames are co-translated as part of acontinuous polypeptide.

As used herein, the term “hemagglutinin tag” or “HA tag” is a proteinderived from a human influenza hemagglutinin surface glycoprotein (HA)corresponding to amino acids 98-106. The HA tag is extensively used as ageneral epitope tag in expression vectors. Recombinant proteins can beengineered to express the HA tag, which does not appear to interferewith the bioactivity or the biodistribution of the recombinant protein.This tag facilitates the detection, isolation, and purification of theprotein of interest.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide from the amino or N-terminus tothe carboxyl or C-terminus, in which amino acids that neighbor eachother in the sequence are contiguous in the primary structure of thepolypeptide.

A portion of a polypeptide that is “amino-terminal” or “N-terminal” toanother portion of a polypeptide is that portion that comes earlier inthe sequential polypeptide chain. Similarly, a portion of a polypeptidethat is “carboxy-terminal” or “C-terminal” to another portion of apolypeptide is that portion that comes later in the sequentialpolypeptide chain.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide that istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

The term “eukaryote” or “eukaryotic organism” is intended to encompassall organisms in the animal, plant, and protist kingdoms, includingprotozoa, fungi, yeasts, green algae, single celled plants, multi celledplants, and all animals, both vertebrates and invertebrates. The termdoes not encompass bacteria or viruses. A “eukaryotic cell” is intendedto encompass a singular “eukaryotic cell” as well as plural “eukaryoticcells,” and comprises cells derived from a eukaryote.

The term “vertebrate” is intended to encompass a singular “vertebrate”as well as plural “vertebrates,” and comprises mammals and birds, aswell as fish, reptiles, and amphibians.

The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited to humans; primatessuch as apes, monkeys, orangutans, and chimpanzees; canids such as dogsand wolves; felids such as cats, lions, and tigers; equids such ashorses, donkeys, and zebras, food animals such as cows, pigs, and sheep;ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears. In certain aspects, the mammal is ahuman subject.

The terms “tissue culture” or “cell culture” or “culture” or “culturing”refer to the maintenance or growth of plant or animal tissue or cells invitro under conditions that allow preservation of cell architecture,preservation of cell function, further differentiation, or all three.“Primary tissue cells” are those taken directly from tissue, i.e., apopulation of cells of the same kind performing the same function in anorganism. Treating such tissue cells with the proteolytic enzymetrypsin, for example, dissociates them into individual primary tissuecells that grow or maintain cell architecture when seeded onto cultureplates. Cell cultures arising from multiplication of primary cells intissue culture are called “secondary cell cultures.” Most secondarycells divide a finite number of times and then die. A few secondarycells, however, can pass through this “crisis period,” after which theyare able to multiply indefinitely to form a continuous “cell line.” Theliquid medium in which cells are cultured is referred to herein as“culture medium” or “culture media.” Culture medium into which desiredmolecules, e.g., viruses or proteins, e.g., immunoglobulin molecules,have been secreted during culture of the cells therein can be referredto as “conditioned medium.”

As used herein, the term “identify” refers to methods in which a desiredmolecule, e.g., a polynucleotide encoding a protein of interest with adesired characteristics or function, is differentiated from a pluralityor library of such molecules. Identification methods include “selection”and “screening” or “panning.” As used herein, “selection” methods arethose in which the desired molecules can be directly separated from thelibrary, e.g., via drug resistance. As used herein, “screening” or“panning” methods are those in which pools comprising the desiredmolecules are subjected to an assay in which the desired molecule can bedetected. Aliquots of the pools in which the molecule is detected arethen divided into successively smaller pools which are likewise assayed,until a pool which is highly enriched from the desired molecule isachieved.

Poxviruses, E.g., Vaccinia Virus EEV Vectors

IMP fusion proteins as provided herein are produced in poxvirus vectors,e.g., vaccinia virus vectors. The term “poxvirus” includes any member ofthe family Poxviridae. See, for example, B. Moss in: Virology, 2dEdition, B. N. Fields, D. M. Knipe et al., Eds., Raven Press, p. 2080(1990). The genus of orthopoxvirus includes, e.g., vaccinia virus,variola virus (the virus that causes smallpox), and raccoon poxvirus.Vaccinia virus is the prototype orthopoxvirus and has been developed andis well-characterized as a vector for the expression of heterologousproteins.

In those embodiments where poxvirus vectors, in particular vacciniavirus vectors, are used to express IMP fusion proteins as providedherein, any suitable poxvirus vector can be used. In certain aspects,the location of a gene encoding an IMP fusion protein can be in a regionof the vector that is non-essential for growth and replication of thevirus so that infectious viruses are produced. Although a variety ofnon-essential regions of the vaccinia virus genome have beencharacterized, the most widely used locus for insertion of foreign genesis the thymidine kinase locus, located in the HindIII J fragment in thegenome. In certain vaccinia virus vectors, the tk locus has beenengineered to contain one or two unique restriction enzyme sites,allowing for convenient use of the trimolecular recombination methodrecombinant virus production, as described elsewhere herein.

Polynucleotides encoding IMP fusion proteins as provided herein can beinserted into poxvirus vectors, particularly vaccinia virus vectors,under operable association with a transcriptional control region whichfunctions in the cytoplasm of a poxvirus-infected cell.

Poxvirus transcriptional control regions comprise a promoter and atranscription termination signal. Gene expression in poxviruses istemporally regulated, and promoters for early, intermediate, and lategenes possess varying structures. Certain poxvirus genes are expressedconstitutively, and promoters for these “early-late” genes bear hybridstructures. Synthetic early-late promoters have also been developed.Suitable poxvirus promoters for expressing IMP fusion proteins asprovided herein include, but are not limited to late promoters such asthe 7.5-kD promoter, the MIL promoter, the 37-kD promoter, the 11-kDpromoter, the 11L promoter, the 12L promoter, the 13L promoter, the 15Lpromoter, the 17L promoter, the 28-kD promoter, the H1L promoter, theH3L promoter, the H5L promoter, the H6L promoter, the H8L promoter, theD11L promoter, the D12L promoter, the D13L promoter, the A1L promoter,the A2L promoter, the A3L promoter, and the P4b promoter. See, e.g.,Moss, B., “Poxviridae and their Replication” IN Virology, 2d Edition, B.N. Fields, D. M. Knipe et al., Eds., Raven Press, p. 2090 (1990).

Suitable poxvirus vectors include wild-type vaccinia virus, e.g., strainWestern Reserve or WR, or attenuated vaccinia virus, e.g., modifiedvaccinia Ankara (MVA) (Mayr, A. et al., Infection 3:6-14 (1975)).

During its replication cycle, a poxvirus, e.g., a vaccinia virus,produces four infectious forms which differ in their membrane structure:intracellular mature virion (IMV), the intracellular enveloped virion(IEV), the cell-associated enveloped virion (CEV) and the extracellularenveloped virion (EEV). The prevailing view is that the IMV have asingle lipoprotein membrane, while the CEV and EEV are both surroundedby two membrane layers and the IEV has three envelopes. EEV is shed fromthe plasma membrane of the host cell and the EEV membrane is derivedfrom the trans-Golgi.

After infection, the virus loses its membrane(s) and the DNA/proteincore is transported along microtubules into the cell. The proteinsencoded by early vaccinia mRNAs (“early” is defined as pre-DNAreplication) lead to uncoating of the vaccinia core and subsequent DNAreplication. This replication occurs in what are termed “viralfactories” which are located essentially on top of the ER. Within theviral factory, immature virions (IV) assemble and are processed to formIMV (Intracellular Mature Virus). IMVs contain a membrane that isderived from the ER. The majority of IMVs are released from the cell bycell lysis. Some IMVs are transported on microtubules to sites ofwrapping by membranes of the trans-Golgi network or early endosomes. Thewrapping of the IMV particles by a double membrane creates a form ofvaccinia called IEVs (Intracellular Enveloped Virus). The IEVs are thentransported to the cell surface on microtubules. The outer IEV membranefuses with the plasma membrane to expose a CEV (Cell AssociatedEnveloped Virus) at the cell surface. Actin polymerization from the hostcell can drive the CEV to infect neighboring cells, or the virus can bereleased as an EEV. See, e.g., Kim L. Roberts and Geoffrey L. Smith.Trends in Microbiology 16 (10):472-479 (2008); Geoffrey L. Smith, etal., Journal of General Virology 83:2915-2931 (2002).

At least six virus-encoded proteins have been reported as components ofthe EEV envelope membrane. Of these, four proteins (A33R, A34R, A56R,and B5R) are glycoproteins, one (A36R) is a nonglycosylatedtransmembrane protein, and one (F13L) is a palmitoylated peripheralmembrane protein. See, e.g., Lorenzo et al., Journal of Virology 74(22):10535 (2000). During infection, these proteins localize to theGolgi complex, where they are incorporated into infectious virus that isthen transported and released into the extracellular medium. As providedherein, IMP fusion proteins are directed to and expressed on the EEVmembrane as a fusion protein with an EEV-specific protein, e.g., F13L orA56R.

The F13L protein is associated with the interior surface of theoutermost EEV membrane through palmitoylation of cysteines 185 and 186.Smith Trends in Microbiol. 16:472-479 (2008). Vaccinia viruses in whichthe gene encoding F13L is deleted form tiny plaques and the number ofEEV produced is reduced significantly.

The amino acid sequence of the F13L protein from vaccinia virus strainWR is presented as SEQ ID NO: 1. The two palmitoylated cysteine residues(amino acids 85 and 86 of SEQ ID NO: 1) are underlined. Since F13L doesnot cross the membrane, it does not have a transmembrane domain orsignal peptide.

>F13L (SEQ ID NO: 1) MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTTRGALIFDKLKEASEKGIKIIVLLDERGKRNLGELQSHCPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGSIHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGVFFTDSPEHLLGYSRDLDTDVVIDKLKSAKTSIDIEHLAIVPTTRVDGNSYYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQNDLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVSEAKKI FERDWVSSHSKSLKI

The A56R protein is the vaccinia virus hemagglutinin and is a standardtype I integral membrane protein comprising an amino-terminalextracellular (“extra-membrane”) domain, a single transmembrane domain,and a cytoplasmic (“intra-membrane”) domain. A56R comprises anN-terminal signal peptide of about 33 amino acids, an Ig-like domainextending from about amino acid 34 to about amino acid 103, a stalkregion extending from about amino acid 121 to about amino acid 275, atransmembrane domain extending from about amino acid 276 to about aminoacid 303, and an cytoplasmic (“inter-membrane”) domain extending fromabout amino acid 304 to amino acid 314. See DeHaven et al., J. GenVirol. 92:1971-1980 (2011). A56R is presented as SEQ ID NO: 5.

>A56R (SEQ ID NO: 5) MTRLPILLLLISLVYATPFPQTSKKIGDDATLSCNRNNTNDYVVMSAWYKEPNSIILLAAKSDVLYFDNYTKDKISYDSPYDDLVTTITIKSLTARDAGTYVCAFFMTSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSR KYKTENKV

IMP fusion proteins as provided herein can be expressed in any suitablevaccinia virus. In certain embodiments, the DNA encoding an EEV fusionprotein can be inserted into a region of the vaccinia virus genome whichis non-essential for growth and replication of the vector so thatinfectious viruses are produced. Although a variety of non-essentialregions of the vaccinia virus genome have been characterized, the mostwidely used locus for insertion of foreign genes is the thymidine kinaselocus, located in the HindIII J fragment in the genome. IMP fusionproteins as provided herein can be inserted into vaccinia virus vectorsunder operable association with a transcriptional control region whichfunctions in the cytoplasm of a poxvirus-infected cell.

Suitable promoters for use in the methods described herein include,without limitation, the early/late 7.5-kD promoter, or the early/late H5promoter (or variants thereof).

The Tri-Molecular Recombination Method

Tri-molecular recombination, as disclosed in Zauderer, PCT PublicationNo. WO 00/028016 and in U.S. Pat. No. 7,858,559, is a high efficiency,high titer-producing method for expressing proteins of interest and orproducing libraries in vaccinia virus. The tri-molecular recombinationmethod allows the generation of recombinant viruses at efficiencies ofat least 90%, and titers at least at least 2 orders of magnitude higherthan those obtained by direct ligation.

In certain aspects, IMP fusion proteins for expression in vaccinia virusand display on EEV as described herein can be constructed in poxvirusvectors, e.g., vaccinia virus vectors, by tri-molecular recombination.

In certain embodiments, a transfer plasmid for IMP fusion proteins forexpression in EEV is provided, which comprises a polynucleotide flankingregions in the vaccinia virus Tk gene, the vaccinia virus H5 promoter,and NcoI and BsiWI restriction sites for inserting coding regions fordesired fusion proteins.

Integral Membrane Proteins

The disclosure provides a method for expressing integral membraneproteins (IMPs) in a conformationally intact state that approaches thenative conformation of the protein as it would appear in a cell in whichthe protein is naturally expressed. According to the disclosure, IMPsare expressed as fusion proteins with poxvirus proteins that areexpressed on poxvirus, e.g., vaccinia virus EEVs. IMP fusion proteins asprovided herein, when expressed and displayed on the surface of EEVs,are useful as target antigens for screening libraries of bindingmolecules, e.g., antibody display libraries.

Any IMP can be constructed as a fusion protein according to the methodsprovided herein. In certain aspects the IMP is a target forimmunotherapy. In certain aspects the IMP is a multi-pass IMP such asCD20 or a G-protein coupled receptor (GPCR). Suitable multi-pass humanIMPs for use in the construction of IMP fusion proteins as providedherein include, without limitation, the proteins listed in Table 1.

TABLE 1 Exemplary Human Multi-Pass Integral Membrane Proteins ENTREZgene # predicted TM Protein Name ENTREZ_gene_ID symbol domainsPoliovirus receptor-related protein 3 25945 PVRL3 2 Prominin-1 8842PROM1 5 FL cytokine receptor 2322 FLT3 2 Scavenger receptorcysteine-rich type 1 9332 CD163 2 protein M130 C-X-C chemokine receptortype 1 3577 CXCR1 6 C-X-C chemokine receptor type 3 2833 CXCR3 7 C-X-Cchemokine receptor type 5 643 CXCR5 7 C-C chemokine receptor type 4 1233CCR4 7 C-C chemokine receptor type 7 1236 CCR7 7 B-lymphocyte antigenCD20 931 MS4A1 4 Major prion protein 5621 PRNP 2 Plexin-C1 10154 PLXNC12 Multidrug resistance protein 1 5243 ABCB1 12 Putative G-proteincoupled receptor 44 11251 GPR44 7 EGF-like module-containing mucin-like30817 EMR2 7 hormone receptor-like 2 Frizzled-4 8322 FZD4 9 Leukocytesurface antigen CD47 961 CD47 5 CD63 antigen 967 CD63 4 Cholinetransporter-like protein 1 23446 SLC44A1 9 CD97 antigen 976 CD97 7Multidrug resistance-associated protein 1 4363 ABCC1 16 CAS1domain-containing protein 1 64921 CASD1 14 Solute carrier family 12member 6 9990 SLC12A6 14 Sodium/hydrogen exchanger 1 6548 SLC9A1 13Solute carrier family 12 member 9 56996 SLC12A9 13 Solute carrier family2, facilitated glucose 6513 SLC2A1 12 transporter member 1 Sodium- andchloride-dependent taurine 6533 SLC6A6 12 transporter Solute carrierorganic anion transporter 28231 SLCO4A1 12 family member 4A1 Solutecarrier family 23 member 2 9962 SLC23A2 12 Solute carrier organic aniontransporter 28232 SLCO3A1 12 family member 3A1 Prestin 375611 SLC26A5 11Equilibrative nucleoside transporter 2 3177 SLC29A2 11 Equilibrativenucleoside transporter 1 2030 SLC29A1 11 Sodium-coupled neutral aminoacid 81539 SLC38A1 11 transporter 1 Sodium bicarbonate cotransporter 39497 SLC4A7 11 Urea transporter 1 6563 SLC14A1 10 Transmembrane andcoiled-coil domain- 55002 TMCO3 10 containing protein 3 Signal peptidepeptidase-like 2A 84888 SPPL2A 9 Transmembrane 9 superfamily member 356889 TM9SF3 9 Anoctamin-9 338440 ANO9 8 Sodium/potassium-transportingATPase 476 ATP1A1 8 subunit alpha-1 Sodium/potassium-transporting ATPase478 ATP1A3 8 subunit alpha-3 Anoctamin-6 196527 ANO6 8 V-type protonATPase 116 kDa subunit a 23545 ATP6V0A2 8 isoform 2 Putative P2Ypurinoceptor 10 27334 P2RY10 7 G-protein coupled receptor 39 2863 GPR397 Sphingosine 1-phosphate receptor 2 9294 S1PR2 7 Latrophilin-2 23266LPHN2 7 Beta-2 adrenergic receptor 154 ADRB2 7 Alpha-2C adrenergicreceptor 152 ADRA2C 7 Thromboxane A2 receptor 6915 TBXA2R 7Platelet-activating factor receptor 5724 PTAFR 7 Proteinase-activatedreceptor 1 2149 F2R 7 Neuropeptide Y receptor type 1 4886 NPY1R 7 Type-1angiotensin II receptor 185 AGTR1 7 Neurotensin receptor type 1 4923NTSR1 7 Cannabinoid receptor 2 1269 CNR2 7 Prostaglandin E2 receptor EP2subtype 5732 PTGER2 7 Calcitonin gene-related peptide type 1 10203CALCRL 7 receptor Protein GPR107 57720 GPR107 7 G-protein coupledreceptor 126 57211 GPR126 7 P2Y purinoceptor 8 286530 P2RY8 7 ProbableG-protein coupled receptor 125 166647 GPR125 7 Transmembrane protein 87A25963 TMEM87A 7 Mas-related G-protein coupled receptor 116535 MRGPRF 7member F Transmembrane protein 87B 84910 TMEM87B 7 Proteinase-activatedreceptor 4 9002 F2RL3 7 Smoothened homolog 6608 SMO 7 EGF-likemodule-containing mucin-like 84658 EMR3 7 hormone receptor-like 3Nemomedin-U receptor 1 10316 NMUR1 7 EGF, latrophilin and seventransmembrane 64123 ELTD1 7 domain-containing protein 1 Transmembraneprotein 8A 58986 TMEM8A 7 Cadherin EGF LAG seven-pass G-type 1952 CELSR27 receptor 2 Cadherin EGF LAG seven-pass G-type 9620 CELSR1 7 receptor 1Cadherin EGF LAG seven-pass G-type 1951 CELSR3 7 receptor 3 Cysteinylleukotriene receptor 1 10800 CYSLTR1 7 G-protein coupled receptor 569289 GPR56 7 Lipid phosphate phosphohydrolase 1 8611 PPAP2A 6 Potassiumvoltage-gated channel subfamily A 3738 KCNA3 6 member 3 Zinc transporterZIP6 25800 SLC39A6 6 Zinc transporter ZIP14 23516 SLC39A14 6 P2Ypurinoceptor 11 5032 P2RY11 6 Zinc transporter ZIP10 57181 SLC39A10 6Cytochrome b-245 heavy chain 1536 CYBB 5 Prominin-2 150696 PROM2 5Protein tweety homolog 2 94015 TTYH2 5 Protein tweety homolog 3 80727TTYH3 5 Gamma-aminobutyric acid receptor subunit 2562 GABRB3 4 beta-3Glutamate receptor, ionotropic kainate 3 2899 GRIK3 4 Neuronal membraneglycoprotein M6-b 2824 GPM6B 4 Metal transporter CNNM4 26504 CNNM4 4Metal transporter CNNM3 26505 CNNM3 3 Discoidin, CUB and LCCL domain-131566 DCBLD2 3 containing protein 2 Transmembrane protein 131-like23240 KIAA0922 2 Leucine-rich repeat transmembrane protein 23768 FLRT2 2FLRT2 Attractin 8455 ATRN 2 Receptor-type tyrosine-protein phosphatase5793 PTPRG 2 gamma Interferon alpha/beta receptor 2 3455 IFNAR2 2 Ephrintype-A receptor 5 2044 EPHA5 2 Tyrosine-protein kinase transmembrane4919 ROR1 2 receptor ROR1 Tomoregulin-1 8577 TMEFF1 2 P2X purinoceptor 75027 P2RX7 2 TM2 domain-containing protein 3 80213 TM2D3 2 TM2domain-containing protein 1 83941 TM2D1 2 G-protein coupled receptor 6410149 GPR64 8 Psychosine receptor 8477 GPR65 6 Large neutral amino acidstransporter small 8140 SLC7A5 12 subunit 1 Sphingosine 1-phosphatereceptor 3 1903 S1PR3 7 Solute carrier organic anion transporter 6578SLCO2A1 12 family member 2A1 Type-2 angiotensin II receptor 186 AGTR2 7UPF0513 transmembrane protein 79583 UNQ870/PRO1886 2 Lipid phosphatephosphohydrolase 3 8613 PPAP2B 5 Blood vessel epicardial substance 11149BVES 3 Sodium/potassium/calcium exchanger 6 80024 SLC24A6 135-hydroxytryptamine receptor 2B 3357 HTR2B 7 Mucolipin-1 57192 MCOLN1 6Cadherin-8 1006 CDH8 2 Adenosine receptor A1 134 ADORA1 7 ProbableG-protein coupled receptor 110 266977 GPR110 7 Chemokine receptor-like 11240 CMKLR1 7 Proton-coupled folate transporter 113235 SLC46A1 11Sphingosine 1-phosphate receptor 4 8698 S1PR4 7 Protein FAM171A2 284069FAM171A2 2 Alpha-2A adrenergic receptor 150 ADRA2A 7 C-X-C chemokinereceptor type 7 57007 CXCR7 7 Apelin receptor 187 APLNR 7 ProbableG-protein coupled receptor 116 221395 GPR116 7 Metalloreductase STEAP479689 STEAP4 6 Solute carrier organic anion transporter 353189 SLCO4C112 family member 4C1 ATP-binding cassette sub-family A member 8 10351ABCA8 14 Vasoactive intestinal polypeptide receptor 1 7433 VIPR1 7 SID1transmembrane family member 2 51092 SIDT2 11 Equilibrative nucleosidetransporter 4 222962 SLC29A4 10 Succinate receptor 1 56670 SUCNR1 7Metal transporter CNNM2 54805 CNNM2 4 Probable palmitoyltransferaseZDHHC5 25921 ZDHHC5 4 Solute carrier family 22 member 16 85413 SLC22A1612 Leukotriene B4 receptor 1 1241 LTB4R 7 Pannexin-1 24145 PANX1 4Sodium-dependent glucose transporter 1 91749 NAGLT1 11 Sodium/calciumexchanger 1 6546 SLC8A1 10 Neuronal acetylcholine receptor subunit 1136CHRNA3 4 alpha-3 Retinoic acid-induced protein 3 9052 GPRC5A 7Lysophosphatidic acid receptor 5 57121 LPAR5 7 Probable G-proteincoupled receptor 132 29933 GPR132 7 Sphingosine 1-phosphate receptor 553637 S1PR5 7 Endothelin-1 receptor 1909 EDNRA 7 Probable G-proteincoupled receptor 124 25960 GPR124 7 Solute carrier family 12 member 710723 SLC12A7 12 Thyrotropin receptor 7253 TSHR 7 Transient receptorpotential cation channel 51393 TRPV2 6 subfamily V member 2 Glutamatereceptor delta-1 subunit 2894 GRID1 4 Gamma-aminobutyric acid receptorsubunit 2555 GABRA2 4 alpha-2 Sphingosine 1-phosphate receptor 1 1901S1PR1 7 Prostaglandin E2 receptor EP3 subtype 5733 PTGER3 7 ProbableG-protein coupled receptor 174 84636 GPR174 7 Glutamate receptor 2 2891GRIA2 3 Amiloride-sensitive sodium channel subunit 6339 SCNN1D 2 delta5-hydroxytryptamine receptor 1D 3352 HTR1D 7 Goliath homolog 55819RNF130 2 ATP-binding cassette sub-family A member 7 10347 ABCA7 11Prostacyclin receptor 5739 PTGIR 7 Probable G-protein coupled receptor176 11245 GPR176 7 Thyrotropin-releasing hormone receptor 7201 TRHR 7Claudin-12 9069 CLDN12 4 Protein FAM38A 9780 FAM38A 29 Niemann-Pick C1protein 4864 NPC1 13 Synaptic vesicle glycoprotein 2A 9900 SV2A 12Signal peptide peptidase-like 2B 56928 SPPL2B 9 Rhomboid family member 279651 RHBDF2 7 Immunoglobulin superfamily member 1 3547 IGSF1 4Dolichyl-diphosphooligosaccharide--protein 6185 RPN2 3glycosyltransferase subunit 2 Transmembrane emp24 domain-containing54732 TMED9 2 protein 9 Steryl-sulfatase 412 STS 2 Transmembrane 9superfamily member 1 10548 TM9SF1 9 Melanoma inhibitory activity protein3 375056 MIA3 2 Arylsulfatase F 416 ARSF 2 Solute carrier family 2,facilitated glucose 6517 SLC2A4 12 transporter member 4 Anoctamin-5203859 ANO5 8 Nicalin 56926 NCLN 2

In certain aspects, the multi-pass IMP is a GPCR, e.g., FZD4 or CXCR4.In certain aspects the multi-pass IMP is CD20.

Polynucleotides Encoding IMP Fusion Proteins for Expression on PoxvirusEEV

This disclosure provides an isolated polynucleotide for expression of anintegral membrane protein or fragment thereof in aconformationally-intact form in the context of a biological membrane, asa fusion with a protein or fragment thereof specific for vaccinia virusEEV. By “conformationally intact” is meant that the protein appears, oris displayed, in a native or close to native conformation in the contextof a biological lipid bilayer membrane, much as the protein would appearin its native state.

In one aspect, the disclosure provides an isolated polynucleotide thatincludes a first nucleic acid fragment that encodes an integral membraneprotein (IMP) or fragment thereof, e.g., a multi-pass IMP, where the IMPor fragment thereof comprises at least one extra-membrane region, atleast one transmembrane domain and at least one intra-membrane region,and where a portion of the first nucleic acid fragment encoding at leastone intra-membrane region is situated at the 5′ or 3′ end of the firstnucleic acid fragment; and a second nucleic acid fragment that encodes avaccinia virus F13L protein (SEQ ID NO: 1) or functional fragmentthereof, where the second nucleic acid fragment is fused in frame to aportion of the first nucleic acid fragment that encodes anintra-membrane region of the IMP. The first nucleic acid fragment andthe second nucleic acid fragment can, in some instances, we separated bya nucleic acid encoding a linker or other spacer. The polynucleotide canfurther include a poxvirus promoter operably associated with the firstand second nucleic acid fragments, allowing expression of thepolynucleotide in the cytoplasm of a poxvirus-infected cell. Accordingto this aspect, a poxvirus-infected cell that contains thepolynucleotide can express an IMP-F13L fusion protein as part of theouter envelope membrane of an extracellular enveloped virion (EEV).Schematic diagrams showing expression of an IMP as a fusion with F13Lare shown in FIG. 1B and FIG. 1C.

In certain aspects, the IMP or fragment thereof can be a multi-passmembrane protein comprising at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, or even moretransmembrane (TM) domains, such as those listed in Table 1.

Where the IMP has an odd number of TM domains, one end of the IMP,either the N-terminus or the C-terminus, will be naturally situated onthe extra-membrane side of the biological membrane and the other end ofthe IMP will be situated on the intra-membrane side of the IMP. Sincethe F13L protein is wholly-internal to the outer membrane of poxvirusEEVs, the end of the IMP, the N-terminus or the C-terminus that issituated internal to the membrane can be fused to F13L. Thus for an IMPsuch as a typical 7-TM domain GPCR in which the N-terminus of theprotein is extra-membrane and the C-terminus is intra-membrane, theN-terminus of F13L can be fused to the C-terminus of the GPCR as shownin FIG. 1B. Accordingly, a polynucleotide as above is provided where thefirst nucleic acid fragment encodes an IMP with an odd number oftransmembrane domains, where the 5′ end of the first nucleic acidfragment encodes the extra-membrane region, and the 3′ end of the firstnucleic acid fragment encodes the intra-membrane region of the IMP, thelatter being fused to the 5′ end of the nucleic acid fragment encodingF13L or a fragment thereof.

In an exemplary polynucleotide of this type, the first polynucleotidecan encode the human frizzled-4 protein (FZD4), or a fragment thereof, atarget for immunotherapy of certain human cancers, fused to theN-terminus of F13L. Accordingly, a polynucleotide which encodes anFZD4-F13L fusion protein is provided. An exemplary polynucleotideaccording to this aspect encodes the mature fusion protein, amino acids20 to 892 of SEQ ID NO: 2, as shown below. The polynucleotide canfurther encode a signal peptide, e.g., the signal peptide of FZD4, aminoacids 1 to 19 of SEQ ID NO: 2.

FZD (FL)-F13L (SEQ ID NO: 2) MGWSCIILFLVATATGAHSFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKEFTDIWMAVWASLCFISTAFTVLTFLIDSSRFSYPERPIIFLSMCYNIYSIAYIVRLTVGRERISCDFEEAAEPVLIQEGLKNTGCAIIFLLMYFFGMASSIWWVILTLTWFLAAGLKWGHEAIEMHSSYFHIAAWAIPAVKTIVILIMRLVDADELTGLCYVGNQNLDALTGFVVAPLFTYLVIGTLFIAAGLVALFKIRSNLQKDGTKTDKLERLMVKIGVFSVLYTVPATCVIACYFYEISNWALFRYSADDSNMAVEMLKIFMSLLVGITSGMWIWSAKTLHTWQKCSNRLVNSGKVKREKRGNGWV KPGKGSETVVMWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTTRGALIFDKLKEASEKGIKIIVLLDERGKRNLGELQSHCPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGSIHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGVFFTDSPEHLLGYSRDLDTDVVIDKLKSAKTSIDIEHLAIVPTTRVDGNSYYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQNDLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVSEAKKIFERDWVSSHSKSLKISingle Underline - leader peptide (amino acids 1-19)Bold - human Fzd4 (amino acids 20-520)Italics - F13L (amino acids 521-892)

In another exemplary polynucleotide of this type, the firstpolynucleotide can encode A CXC chemokine receptor, or a fragmentthereof, fused to the N-terminus of F13L. CXC chemokine receptors arelikewise targets for immunotherapy of certain human cancers. Anexemplary CXC chemokine receptor is CXCR4, or a fragment thereof.Accordingly, a polynucleotide which encodes a CXC chemokinereceptor-F13L fusion protein, e.g., a CXCR4-F13L fusion protein isprovided. An exemplary polynucleotide according to this aspect encodesSEQ ID NO: 3, as shown below.

CXCR4-F13L (SEQ ID NO: 3)MAIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTTRGALIFDKLKEASEKGIKIIVLLDERGKRNLGELQSHCPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGSIHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGVFFTDSPEHLLGYSRDLDTDVVIDKLKSAKTSIDIEHLAIVPTTRVDGNSYYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQNDLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVSEAKKIF ERDWVSSHSKSLKIBold - human CXCR4 (amino acids 1-356)Italics - F13L (amino acids 357-728)

As will be evident to a person of ordinary skill in the art, amulti-pass membrane protein having an even number of transmembranedomains will be inserted into a biological membrane such that itsN-terminus and its C-terminus are on the same side of the membrane,either on the extra-membrane side of the membrane, or on theintra-membrane side of the membrane. Since the F13L protein is situatedentirely on the intra-membrane side of poxvirus EEVs, formation of anIMP-F13L fusion protein properly embedded in the membrane would need atleast one of the N-terminus or the C-terminus of the IMP or fragmentthereof to be internal to the membrane. Where the IMP has an even numberof TM domains and both are situated internally, the F13L protein can befused either to the N-terminus of the IMP or to the C-terminus of theIMP. If the full-length IMP is situated such that both the N- andC-terminus are extra-membrane, a fragment of the IMP having an oddnumber of TM domains can be fused to F13L.

Accordingly, the disclosure provides a polynucleotide as described abovethat encodes an IMP with an even number of transmembrane domains, whereboth the 5′ and 3′ ends of the first nucleic acid fragment encodeintra-membrane regions. In certain aspects the 3′ end of the nucleicacid fragment encoding F13L can be fused to the 5′ end of the nucleicacid fragment encoding the IMP, in certain aspects the 5′ end of thenucleic acid fragment encoding F13L can be fused to the 3′ end of thenucleic acid fragment encoding the IMP.

An exemplary IMP of this type is human CD20, a 4-TM domain IMP expressedon human B cells, which is a target for immunotherapy of B cellleukemias, lymphomas, and myelomas. A diagram of a CD20-F13L fusionprotein in which the C-terminus of CD20 is fused to the N-terminus ofF13L is shown in FIG. 1C. Accordingly, a polynucleotide which encodes aCD20-F13L fusion protein is provided. An exemplary polynucleotideaccording to this aspect encodes SEQ ID NO: 4, as shown below.

CD20-F13L (Seq ID NO: 4)MATPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTTRGALIFDKLKEASEKGIKIIVLLDERGKRNLGELQSHCPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGSIHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGVFFTDSPEHLLGYSRDLDTDVVIDKLKSAKTSIDIEHLAIVPTTRVDGNSYYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQNDLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVSEAKKIFERDWVSSH SKSLKIBold - human CD20 (MS4A1) (amino acids 1-298)Italics - F13L (amino acids 299-669)

In polynucleotides as provided above, the first and second nucleic acidfragments can be directly fused, or alternatively they can be separatedby a nucleic acid fragment encoding a linker or spacer or otherpolypeptide fragment. In certain aspects, a polynucleotide as providedabove can further include a third nucleic acid fragment that encodes aheterologous peptide polypeptide, either between the first and secondnucleic acid fragments, or on either side. The heterologous peptide canbe, for example, a linker sequence, an amino acid tag or label, or apeptide or polypeptide sequence that facilitates purification. Incertain aspects the heterologous peptide is a 6-histidine tag fused,e.g., to the C-terminus of the fusion protein.

In certain aspects, a polynucleotide as provided herein is operablyassociated with a poxvirus promoter. Suitable promoters are describedelsewhere herein. In certain aspects the promoter is a poxvirus p7.5promoter or a poxvirus H5 promoter.

A polynucleotide as provided herein can be or can be part of, a poxvirusgenome, where the poxvirus genome, upon introduction into a suitablepermissive host cell, can produce infectious EEV that display theIMP-F13L fusion protein on their surface. In certain aspects thepoxvirus genome is a vaccinia virus genome, e.g., a vaccinia virus WRgenome or an MVA genome. A poxvirus genome comprising a polynucleotideas described can be produced by standard molecular biological andvirology techniques, for example by using tri-molecular recombination asdescribed herein. A poxvirus genome as provided herein can be introducedinto permissive cells as part of a recombinant poxvirus, or as naked DNAaccompanied by suitable helper viruses, e.g., fowlpox virus. Thedisclosure further provides a recombinant poxvirus, e.g., a recombinantvaccinia virus comprising the provided poxvirus genome.

IMP-EEV Fusion Proteins, Recombinant Poxvirus EEVs, and Methods ofMaking

This disclosure further provides an IMP-F13L fusion protein such asthose encoded by the polynucleotides described above. Moreover, theIMP-F13L fusion protein can be expressed on the surface of a recombinantpoxvirus EEV, e.g., a recombinant vaccinia virus EEV. A recombinantpoxvirus EEV, e.g., a recombinant vaccinia virus EEV comprising theprovided fusion protein is provided by the disclosure. A recombinantpoxvirus EEV can be produced by a method that includes infecting a hostcell permissive for vaccinia virus infectivity with a vaccinia viruscomprising a poxvirus genome as provided above and recovering EEVreleased from the infected host cell. Accordingly, an IMP-F13L fusionprotein encoded by a polynucleotide as described above, is provided.

Moreover the disclosure provides fusion proteins comprising an IMP orfragment thereof, which can be a multi-pass IMP, and single pass IMP, oreven just the extracellular domain (ECD) of the IMP, fused to a poxvirusprotein, e.g., a vaccinia virus protein, specific for EEV, such as F13L,A56R, B5R, 33R, A34R, or A36R, an “IMP-EEV fusion protein.” ExemplaryECD fusion proteins are described below. An IMP-EEV fusion protein asprovided herein can display the IMP, e.g., a multi-pass IMP, single-passIMP or ECD of an IMP, in a conformationally intact form on the surfaceof poxvirus EEV. For use in screening antibody display libraries forantigen binding domains that specifically bind to a target IMP, displayof IMPs on the surface of poxvirus EEV offers many advantages overdisplaying IMPs on the surface of recombinant cells, e.g., CHO cells, asis typical. For example, the IMP can be expressed at higher density onEEV than on cells. Moreover, EEV express only about six differentpoxvirus proteins on their surface (e.g., F13L, A56R, B5R, 33R, A34R,and A36R) as opposed to hundreds that might be expressed on the surfaceof cells. Finally, inactivated EEV expressing IMP-F13L fusion proteinsas provided herein can be attached to solid supports, offeringconvenience in library screening.

Accordingly, this disclosure provides a method to display an integralmembrane protein (IMP) or fragment thereof in a native conformation foruse, e.g., in screening antibody display libraries for antigen bindingdomains specific for the IMP. The method includes: infecting host cellspermissive for poxvirus infectivity with a recombinant poxvirus thatexpresses the IMP or fragment thereof as a fusion protein with poxvirusEEV-specific protein or membrane-associated fragment thereof, where EEVproduced by the infected host cell comprise the IMP as part of the EEVouter envelope membrane; and recovering EEV released from the host cell.IMP. In certain aspects, the EEV-specific protein or fragment thereofcan be the vaccinia virus A33R protein, A34R protein, A56R protein, B5Rprotein, A36R protein, F13L protein, any membrane-associated fragmentthereof, or any combination thereof.

In certain aspects, the EEV-specific protein is F13L (SEQ ID NO: 1) or afunctional fragment thereof, and the fusion protein can be one expressedby a polynucleotide as provided above, e.g., where the IMP is amulti-pass membrane protein comprising at least two, at least three, atleast four, at least five, at least six or at least seven transmembranedomains.

In certain aspects, the membrane-associated EEV specific proteinfragment includes the stalk, transmembrane, and intra-membrane domainsof the vaccinia virus A56R protein, a fragment comprising, consistingof, or consisting essentially of amino acids 108 to 314 of SEQ ID NO: 5.One of several exemplary fusion partners includes the ECD of human FZD4,shown in bold in SEQ ID NO: 6 below. According to this exemplary aspectthe disclosure provides a method to display a conformationally intactfragment of human FZD4 on the surface of a poxvirus EEV comprisinginfecting host cells permissive for poxvirus infectivity with arecombinant poxvirus encoding a fusion protein comprising amino acids 20to 370 of SEQ ID NO: 6. In certain aspects the fusion protein canfurther comprise a signal peptide, e.g., amino acids 1 to 19 of SEQ IDNO: 6.

FZD-ECD-A56R (Seq ID NO: 6) MGWSCIILFLVATATGAHSFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLP HKTPIQPGEETSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYTY NKRSRKYKTENKV.Single Underline - leader peptide (amino acids 1-19)Bold - human FZD4 extracellular domain (amino acids 20-163)Italics - A56R stalk, transmembrane, and intra-membrane (amino acids 164 to 370)

Another exemplary fusion partner includes the ECD of human ErbB2 (Her2),shown in bold in SEQ ID NO: 7 below. According to this exemplary aspectthe disclosure provides a method to display a conformationally intactfragment of human Her2 on the surface of a poxvirus EEV comprisinginfecting host cells permissive for poxvirus infectivity with arecombinant poxvirus encoding a fusion protein comprising amino acids 20to 855 of SEQ ID NO: 7. In certain aspects the fusion protein canfurther comprise a signal peptide, e.g., amino acids 1 to 19 of SEQ IDNO: 7.

Her2-A56R (SEQ ID NO: 7) MGWSCIILFLVATATGAHSSTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASP TSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRKYKTENKV.Single Underline - leader peptide (amino acids 1-19)Bold - human ERBB2 (HER2) extracellular domain (amino acids 20-648)Italics - A56R stalk, transmembrane, and intra-membrane (amino acids 649 to 855)

Another exemplary fusion partner includes the ECD of human CD100(Semaphorin 4D), shown in bold in SEQ ID NO: 8 below. According to thisexemplary aspect the disclosure provides a method to display aconformationally intact fragment of human CD100 on the surface of apoxvirus EEV comprising infecting host cells permissive for poxvirusinfectivity with a recombinant poxvirus encoding a fusion proteincomprising amino acids 20 to 935 of SEQ ID NO: 8. In certain aspects thefusion protein can further comprise a signal peptide, e.g., amino acids1 to 19 of SEQ ID NO: 8.

CD100-A56R (SEQ ID NO: 8) MGWSCIILFLVATATGAHSFAPIPRITWEHREVHLVQFHEPDIYNYSALLLSEDKDTLYIGAREAVFAVNALNISEKQHEVYWKVSEDKKAKCAEKGKSKQTECLNYIRVLQPLSATSLYVCGTNAFQPACDHLNLTSFKFLGKNEDGKGRCPFDPAHSYTSVMVDGELYSGTSYNFLGSEPIISRNSSHSPLRTEYAIPWLNEPSFVFADVIRKSPDSPDGEDDRVYFFFTEVSVEYEFVFRVLIPRIARVCKGDQGGLRTLQKKWTSFLKARLICSRPDSGLVFNVLRDVFVLRSPGLKVPVFYALFTPQLNNVGLSAVCAYNLSTAEEVFSHGKYMQSTTVEQSHTKWVRYNGPVPKPRPGACIDSEARAANYTSSLNLPDKTLQFVKDHPLMDDSVTPIDNRPRLIKKDVNYTQIVVDRTQALDGTVYDVMFVSTDRGALHKAISLEHAVHIIEETQLFQDFEPVQTLLLSSKKGNRFVYAGSNSGVVQAPLAFCGKHGTCEDCVLARDPYCAWSPPTATCVALHQTESPSRGLIQEMSGDASVCPDKSKGSYRQHFFKHGGTAELKCSQKSNLARVFWKFQNGVLKAESPKYGLMGRKNLLIFNLSEGDSGVYQCLSEERVKNKTVFQVVAKHVLEVKVVPKPVVAPTLSVVQTEGSRIATKVLVASTQGSSPPTPAVQATSSGAITLPPKPAPTGTSCEPKIVINTVP QLHSEKTMYLKSSDTSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCIT YYIYNKRSRKYKTENKV.Single Underline - leader peptide (amino acids 1- 19)Bold - human CD100 extracellular domain (amino acids 20-728)Italics - A56R stalk, transmembrane, and intra-membrane (amino acids 729 to 935)

In certain aspects, the membrane-associated EEV specific proteinfragment includes the transmembrane and intra-membrane domains of thevaccinia virus B5R protein, a fragment comprising, consisting of, orconsisting essentially of amino acids 276 to 317 of SEQ ID NO: 9. Incertain aspects, the membrane-associated EEV specific protein fragmentincludes the stalk, transmembrane, and intra-membrane domains of thevaccinia virus B5R protein, a fragment comprising, consisting of, orconsisting essentially of amino acids 238 to 317 of SEQ ID NO: 9.

SEQ ID NO: 9: WR B5R proteinMKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDQGYHSSDPNAVCETDKWKYENPCKKMCTVSDYISELYNKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGASYISCTANSWNVIPSCQQKCDMPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPVLPICVRTNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIVALTIMGVIFLISVIVLVCSCDKN NDQYKFHKLLP

In certain exemplary aspects the IMP fusion partner for the B5R fragmentcomprises the extracellular domain of human FZD4, shown in bold in SEQID NO: 10 and SEQ ID NO: 11 below. According to this exemplary aspectthe disclosure provides a method to display a conformationally intactfragment of human FZD4 on the surface of a poxvirus EEV comprisinginfecting host cells permissive for poxvirus infectivity with arecombinant poxvirus encoding a fusion protein comprising amino acids 20to 243 of SEQ ID NO: 10 or amino acids 20 to 281 of SEQ ID NO: 11. Incertain aspects the fusion protein can further comprise a signalpeptide, e.g., amino acids 1 to 19 of SEQ ID NO: 10.

FZD-B5R (short) (SEQ ID NO: 10) MGWSCIILFLVATATGAYAFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKEC ATYHIIIVALTIMGVIFLISVIVLVCSCDKNNDQYKFHKLLP.Single Underline - leader peptide (amino acids 1- 19)Bold - human FZD4 extracellular domain (amino acids 20-200)Italics - B5R TM and cytoplasmic tail (amino acids 201-243)FZD-B5R (long) (SEQ ID NO: 11) MGWSCIILFLVATATGAYAFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKE YVRTNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIVALTIMGVIFLISVIVLVCSCDKNNDQYKEHKLLP.Single Underline - leader peptide (amino acids 1- 19)Bold - human FZD4 extracellular domain (amino acids 20-200)Italics - B5R stalk, TM and cytoplasmic tail (amino acids 201-281)

The disclosure further provides a fusion protein comprising: amino acids20 to 892 of SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; amino acids 20 to370 of SEQ ID NO: 6; amino acids 20 to 855 of SEQ ID NO: 7; amino acids20 to 935 of SEQ ID NO: 8; amino acids 20 to 243 of SEQ ID NO: 10; oramino acids 20 to 281 of SEQ ID NO: 11, any combination thereof, anyfragment thereof, or any variant thereof, where the fusion protein, whenexpressed by a recombinant poxvirus, appears on the surface of apoxvirus extracellular enveloped virion (EEV) in a native conformation.

A recombinant poxvirus EEV comprising any EEV fusion protein as providedherein is also provided.

Method of Selecting Antibodies

This disclosure further provides a method to select binding molecules,e.g., antibodies, antigen-binding antibody fragments, or antibody likebinding molecules that bind to a multi-pass membrane protein interest.The method comprises attaching a recombinant EEV as provided herein to asolid support, where the recombinant EEV can display a multi-passprotein on its surface; providing a display library, e.g., an antibodydisplay library, where the library comprises display packages displayinga plurality of antigen binding domains; contacting the display librarywith the EEV such that display packages displaying antigen bindingdomains that specifically binds to the IMP expressed on the EEV can bindthereto; removing unbound display packages; and recovering displaypackages that display an antigen binding domain specific for the IMPexpressed on the EEV.

Any display library comprising a plurality of binding domains, e.g.,antibodies, antibody like molecules or other binding molecules issuitable for this method. For example the display library can be a phagedisplay library, a yeast display library or a library constructed in avaccinia virus vector as described elsewhere herein.

In certain aspects, the recombinant EEV can be inactivated prior toattachment to the solid support. For example, the EEV can be inactivatedby incubation with Psoralen (Trioxsalen, 4′-aminomethyl-, hydrochloride)in the presence of UV irradiation.

Any suitable solid support can be used. As used herein, a “solidsupport” is any support capable of binding an EEV, which can be in anyof various forms, as is known in the art. Well-known supports includetissue culture plastic, glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thisdisclosure. The support material can have virtually any structuralconfiguration as long as the coupled EEV is capable of binding to adisplayed binding molecule such as an antibody. Thus, the supportconfiguration can be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface can be flat such as a sheet, test strip, etc.Typical supports include beads, e.g., magnetic polystyrene beads such asDYNABEADS® that can be pulled out of suspension by a magnet. The supportconfiguration can include a tube, bead, microbead, well, plate, tissueculture plate, petri plate, microplate, microtiter plate, flask, stick,strip, vial, paddle, etc., etc. A solid support can be magnetic ornon-magnetic. Those skilled in the art will know many other suitablecarriers for binding EEV as provided herein, or will be able to readilyascertain the same. In certain aspects, EEV as provided herein can beattached to the solid support via reaction with, e.g., tosyl groups,epoxy groups, carboxylic acid groups, or amino groups attached to thesurface. For example, EEV can be attached to the surface oftosyl-activated magnetic beads, e.g., MYONE™ tosylactivated beads.Alternatively, the EEV can be biotinylated and attached to astreptavidin solid surface, e.g., streptavidin coated magnetic beads.

This disclosure employs, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. (See, for example, Sambrook et al., ed. (1989) MolecularCloning A Laboratory Manual (2nd ed.; Cold Spring Harbor LaboratoryPress); Sambrook et al., ed. (1992) Molecular Cloning: A LaboratoryManual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985)DNA Cloning, Volumes I and II; Gait, ed. (1984) OligonucleotideSynthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins,eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984)Transcription And Translation; Freshney (1987) Culture Of Animal Cells(Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986);Perbal (1984) A Practical Guide To Molecular Cloning; the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Caloseds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold SpringHarbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In CellAnd Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described can be followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freeman & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlag); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties. Thefollowing examples are offered by way of illustration and not by way oflimitation.

EXAMPLES Example 1: Fusion Protein Construction

IMPs were incorporated into vaccinia virus EEVs using the EEV-specificproteins F13L, A56R, and B5R, by the following methods. Generally, theextracellular domains of HER2, CD100 (semaphorin 4D), and FZD4 wereincorporated as fusions with the single-pass EEV-specific membraneproteins A56R and B5R as diagrammed in FIG. 1A. The mature FZD4-ECD-A56Rfusion protein comprises amino acids 20 to 370 of SEQ ID NO: 6, themature HER2-ECD-A56R fusion protein comprises amino acids 20 to 855 ofSEQ ID NO: 7, and the mature CD100-ECD-A56R fusion protein comprisesamino acids 20 to 935 SEQ ID NO: 8. FIG. 1B and FIG. 1C showdiagrammatically how the multi-pass proteins such as GPCRs and CD20 canbe incorporated into EEVs as multi-pass membrane proteins as a fusionwith the EEV membrane-associated protein F13L.

Preparation of F13L Fusion Proteins (FZD4-F13L, CD20-F13L, andCXCR4-F13L)

cDNAs encoding the IMPs were cloned in-frame to the vaccinia virus F13Lgene encoding the palmitoylated EEV membrane glycoprotein (SEQ ID NO: 1)into the pJEM1 plasmid previously described for the purpose ofintroduction into vaccinia virus. pJEM1 is a derivative of p7.5/tkdescribed in U.S. Patent Appl. Publ. No. 2013/0288927, and when digestedwith NcoI or BssHII and BsiWI, contains flanking regions capable ofhomologous recombination with the vaccinia virus TK gene and thevaccinia virus H5 promoter.

The open reading frame of human membrane protein MS4A1 gene(CD20)(NM_021950.3) was cloned in frame with the vaccinia virus F13Lusing SOE (Splicing by Overlap Extension) PCR as per standard protocolswhereby restriction endonuclease sites NcoI and BsiWI were added to thePCR product by encoding them into the 5′ and 3′-most flanking primersrespectively. This strategy avoids the introduction of a leader peptide.The final PCR product and pJEM1 were digested with NcoI and BsiWI andthe two species were joined via ligation according to standardprotocols. The circularized plasmid was then introduced into competentE. coli via chemical transformation and colonies were selected onampicillin-containing agar plates.

MS4A15 (SEQ ID NO: 12) tataCCATGgCAACACCCAGAAATTCAGTAAATG MS4A1AS(SEQ ID NO: 13) GGTACCGATGCAAATGGCCACATAGGAGAGCTGTCATTTTCTATTGG F135(SEQ ID NO: 14) CCAATAGAAAATGACAGCTCTCCTATGTGGCCATTTGCATCGGTACC F13AS(SEQ ID NO: 15) tataCGTACGTTAATGGTGATGGTGATGATGAATTTTTAACGATTTACTGT G

The resulting CD20-F13L fusion protein encoded by the polynucleotidecomprises the amino acid sequence SEQ ID NO: 4.

The open reading frame of human membrane protein FZD4 (NM_012193.3) wascloned in frame with the vaccinia virus F13L using SOE (Splicing byOverlap Extension) PCR as per standard protocols whereby restrictionendonuclease sites BssHII and BsiWI were added to the PCR product byencoding them into the 5′ and 3′-most flanking primers, respectively.This strategy provides for the use of the leader peptide containedwithin pJEM1. The final PCR product and pJEM1 were digested with BssHIIand BsiWI and the two species were joined via ligation according to thestandard protocols. The circularized plasmid was then introduced intocompetent E. coli via chemical transformation and colonies were selectedon ampicillin-containing agar plates. PCR primers were specific for FZD4and F13L and conform to the same general strategy as described forMS4A1. The resulting mature FZD4-F13L fusion protein encoded by thepolynucleotide comprises amino acids 20-892 of SEQ ID NO: 2.

The open reading frame of human membrane protein CXCR4 (NM_001008540.1)was cloned in frame with the vaccinia virus F13L using SOE (Splicing byOverlap Extension) PCR as per standard protocols whereby restrictionendonuclease sites NcoI and BsiWI were added to the PCR product byencoding them into the 5′ and 3′-most flanking primers respectively.This strategy avoids the introduction of a leader peptide. The final PCRproduct and pJEM1 were digested with NcoI and BsiWI and the two specieswere joined via ligation according to the standard protocol. Thecircularized plasmid was then introduced into competent E. coli viachemical transformation and colonies were selected onampicillin-containing agar plates. PCR primers were specific for CXCR4and F13L and conform to the same general strategy as described forMS4A1. The resulting CXCR4-F13L fusion protein encoded by thepolynucleotide comprises the amino acid sequence SEQ ID NO: 3.

The plasmids produced as described above, as well as similar plasmidsencoding non-fused (“untagged”) versions of CD20, FZD4, CXCR4, CD100,and HER2, were linearized and introduced into vaccinia virus viatri-molecular recombination.

Example 2: Expression of CD20-F13L Fusion Protein on EEV

BHK cells were infected with either IMV encoding the CD20-F13L fusionprotein (SEQ ID NO: 4) or Control Western Reserve (WR) virus at amultiplicity of infection (MOI) of 1 virus per cell for two days afterwhich the supernatant containing EEV was harvested and debris removed bylow speed centrifugation. Protein G DYNABEADS® (110 μL) were pulled downwith a magnet and 1 mL of PBS+20 μg of purified anti-CD20 antibody wasadded to the beads. The solution was incubated at room temperature withgentle rotation for 30-60 minutes to allow the antibody to couple to theProtein G beads. Ten μg of purified mIgG1 isotype control was added tothe solution to ensure complete blocking, and the solution was incubatedat room temperature with gentle rotation for 10-30 additional minutes.Beads were pulled down with the magnet, washed once with 1 mL of PBS andresuspended in 110 μL of PBS.

Fifty μL of Anti-CD20-Pro G DYNABEADS® was added to 1 mL of CD20-F13L orWR EEV supernatant and was incubated at room temperature with gentlerotation for 1 hour. Beads were pelleted using the magnet and unboundsupernatant removed. The beads were then washed five times with 1 mL ofDulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% FBSand 1 mM HEPES (10% DMEM). All washes were pooled with the unboundsupernatant (“Unbound”). The beads (“Bound”) were then resuspended in 1mL of 10% DMEM. “Unbound” and “Bound” were titered on BSC-1 cells andoverlaid with growth medium containing methylcellulose. Plaques wereallowed to form for two days and then the cells were fixed and stainedwith 0.1% Crystal Violet solution. Plaques were counted to determine thenumber of plaque forming units (pfu) in the “Unbound” and “Bound” fromwhich the % of EEV bound to the beads could be calculated. Results areshown on Table 2.

TABLE 2 CD20-F13L EEV Binding EEV Supernatant % Bound Western Reserve10.8% CD20-F13L 50.5%

The % EEV bound to the anti-CD20 coated beads was significantly higherfor CD20-F13L EEV fusion protein than it is for the Western Reserveindicating that CD20 is being expressed on the EEV membrane surface.

Example 3: Fusion of CD20 to F13L is More Efficiently Expressed on theEEV Membrane

BHK cells were infected with either IMV encoding the CD20-F13L fusionprotein (SEQ ID NO: 4), CD20-A56R fusion protein (SEQ ID NO: 16), CD20untagged (unfused) or control HER2-A56R Extracellular Domain (ECD) (SEQID NO: 7) virus at a MOI=1 for two days after which the supernatantcontaining EEV was harvested and debris removed by low speedcentrifugation. Streptavidin DYNABEADS® (200 μL) were pulled down with amagnet and 0.2 mL of PBS+20 μg of purified Biotinylated anti-CD20antibody or Biotinylated anti-HER2 was added to the beads. The solutionwas incubated at room temperature with gentle rotation for 30 minutes toallow the antibody to couple to the Streptavidin beads. Beads werepulled down with the magnet, washed once with 1 mL of PBS andresuspended in 200 μL of PBS.

CD20-A56R: (SEQ ID NO: 16) MGWSCHLFLVATATGAHTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRKYKT ENKV

TFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIVAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSPSingle Underline - Signal sequence (amino acids 1- 19)Italics - Truncated A56R (amino acids 20-190)Bold - CD20 Sequence (amino acids 191-506)

Fifty μL of prepared streptavidin beads were added to 1 mL of each EEVsupernatant and allowed to rotate at room temperature for 45 minutes.Beads were pelleted using the magnet and unbound supernatant removed.The beads were then washed five times with 1 mL of DMEM mediasupplemented with 10% FBS and 1 mM HEPES (10% DMEM). All washes werepooled with the unbound supernatant (“Unbound”). The beads (“Bound”)were then resuspended in 1 mL of 10% DMEM. “Unbound” and “Bound” weretitered on BSC-1 cells and overlaid with growth medium containingmethylcellulose. Plaques were allowed to form for two days and then thecells were fixed and stained with 0.1% Crystal Violet solution. Plaqueswere counted to determine the number of plaque forming units (pfu) inthe “Unbound” and “Bound” from which the % of EEV bound to the beadscould be calculated. Results are shown in FIG. 2.

The % EEV bound to the anti-CD20 coated beads for CD20-F13L was greaterthan the % EEV bound for untagged (unfused) or A56R-fused CD20indicating higher expression of CD20-F13L on the EEV membrane. The lackof binding to the anti-HER2 coated beads confirmed specificity of theassay.

The experiment above was repeated using CD20-F13L fusion protein (SEQ IDNO: 4), CD20 untagged (unfused), FZD-F13L fusion protein (SEQ ID NO: 2),and FZD untagged (unfused). Virus was pulled down using anti-CD20 oranti-FZD coated beads as described above. The data in FIG. 3A(anti-CD20-coated beads) and FIG. 3B (anti-FZD-coated beads) shows thatF13L fusion proteins were specifically pulled down by their respectiveantibodies and were more efficiently incorporated into vaccinia virusthan untagged (unfused) proteins.

Example 4: Vaccinia Virus can be engineered to Express VariousAntigen-EEV Constructs

BHK cells were infected at a MOI=1 with virus expressing the followingantigen constructs: CD20-F13L (SEQ ID NO: 4), CXCR4-F13L (SEQ ID NO: 3),HER2-ECD-A56R (SEQ ID NO: 7), and CD100-ECD-A56R (SEQ ID NO: 8). Aftertwo days, the supernatant containing EEV was harvested and debrisremoved by low speed centrifugation. Streptavidin DYNABEADS® were pulleddown with a magnet and for each sample, 50 μL of beads were resuspendedin 0.1 mL of PBS+5 μg of purified Biotinylated anti-CD20 antibody,Biotinylated anti-CXCR4 (12G5), Biotinylated anti-CD100 (2503), orBiotinylated anti-HER2. The solutions were incubated at room temperaturewith gentle rotation for 30 minutes to allow the antibody to couple tothe Streptavidin beads. Beads were pulled down with the magnet, washedonce with 1 mL of PBS and resuspended in 100 μL of PBS per sample.

One hundred μL of prepared streptavidin beads were added to 1 mL of eachEEV supernatant and allowed to rotate at room temperature for 45minutes. Beads were pelleted using the magnet and unbound supernatantremoved. The beads were then washed five times with 1 mL of DMEM mediasupplemented with 10% FBS and 1 mM HEPES (10% DMEM). All washes werepooled with the unbound supernatant (“Unbound”). The beads (“Bound”)were then resuspended in 1 mL of 10% DMEM. “Unbound” and “Bound” weretitered on BSC-1 cells and overlaid with growth medium containingmethylcellulose. Plaques were allowed to form for two days and then thecells were fixed and stained with 0.1% Crystal Violet solution. Plaqueswere counted to determine the number of plaque forming units (pfu) inthe “Unbound” and “Bound” from which the % of EEV bound to the beadscould be calculated. Results are shown in FIG. 4.

All of the antigen-EEV bound specifically to their correspondingantibody-coupled beads indicating efficient expression of the antigen onthe EEV membrane.

Example 5: Antigen-EEV can be Directly Coupled to Magnetic Beads forAntibody Selection

BHK cells (2×10⁸ cells) were infected at a MOI=1 with virus expressingHER2-ECD-A56R (SEQ ID NO: 7), FZD-F13L (SEQ ID NO: 2), CXCR4-F13L (SEQID NO: 3) or CD100 (semaphorin 4D)-ECD-A56R (SEQ ID NO: 8) in onecellSTACK cell culture chamber each (Corning). After two days, thesupernatant containing EEV was harvested and debris removed by low speedcentrifugation. The clarified supernatant was then spun at 13,000 rpm(28,000×g) for 1 hour to pellet the antigen-EEV. The supernatant wasaspirated and the pellet resuspended in 1.5 mL of 1× PBS. The variousviruses were transferred to fresh tubes and Psoralen (Trioxsalen,4′-aminomethyl-, hydrochloride; Sigma) was added to 20 μg/ml finalconcentration. The EEV and Psoralen were incubated at room temperaturefor 10 minutes before being irradiated in the STRATALINKER® UVCrosslinker (Stratagene) for 99,999 microjoules. The Psoralen/UVprocedure ensures that the antigen-EEV is inactivated and thereforeunable to form plaques or multiply in any downstream testing.

Tosylactivated MyOne DYNABEADS® (100 μL) were pulled down with a magnetand washed with 1 mL of PBS. The beads were pulled down with the magnet,the PBS removed and 1 mL of each Psoralen/UV inactivated antigen-EEV wasadded to a separate aliquot of beads. The beads and antigen-EEV wereallowed to rotate at 37° C. for 16-20 hours. The beads were pelleted andthe supernatant was removed. The beads were blocked with 1 mL of 1× PBS,10% FBS and 0.5% BSA at 37° C. for 1 hour. The beads were pelleted andwashed with 1 mL 1× PBS before being resuspended in 200 μL of 1× PBS.

One hundred microliters of antigen-EEV-coupled beads was added to 1 mLof each respective antibody-EEV supernatant expressing anti-FZD4,anti-CXCR4, anti-CD100, or anti-HER2, as well as to controlantibody-EEV. Antibody EEV were produced by infecting BHK cells at aMOI=1 each for 2 days with vaccinia virus encoding both the heavy andlight chains of the respective antibodies, and harvesting thesupernatants followed by a low speed spin to remove any cells. The viruscoupled beads and antibody EEV were allowed to rotate at roomtemperature for 2 hours. Beads were pelleted using the magnet andunbound supernatants removed. The beads were then washed five times with1 mL of DMEM media supplemented with 10% FBS and 1 mM HEPES (10% DMEM).All washes were pooled with the unbound supernatant (“Unbound”). Thebeads (“Bound”) were then resuspended in 1 mL of 10% DMEM. “Unbound” and“Bound” were titered on BSC-1 cells and overlaid with growth mediumcontaining methylcellulose. Plaques were allowed to form for two daysand then the cells were fixed and stained with 0.1% Crystal Violetsolution. Plaques were counted to determine the number of plaque formingunits (pfu) in the “Unbound” and “Bound” from which the % of EEV boundto the beads could be calculated. A diagram of the method is shown inFIG. 5, and results are shown in FIG. 6A (HER2), FIG. 6B (FZD4), FIG. 6C(CXCR4), and FIG. 6D (CD100 (“Sema”)).

Antibody-EEV expressing Anti-HER2 was specifically pulled down by beadscoupled with HER2-ECD-A56R antigen EEV, Antibody-EEV expressing Anti-FZDwas specifically pulled down by beads coupled with FZD-F13L antigen EEV,Antibody-EEV expressing Anti-CXCR4 was specifically pulled down by beadscoupled with CXCR4-F13L antigen EEV, and Antibody-EEV expressingAnti-SEMA was specifically pulled down by beads coupled withSema-ECD-A56R antigen EEV.

Example 6: Antibody Library Screening

BHK cells were infected at a MOI=1 each with an antibody library(H-IgG-A56R) and L48 (derivative of germline VK1-39) in four cellSTACKcell culture chamber (Corning) (2×10⁸ cells per stacker). The antibodylibrary contained a diverse population of heavy chain variable domainsin full length IgG format, fused in frame to A56R (see US Patent Appl.Publication No. 2013-0288927, which is incorporated herein by referencein its entirety). The diversity of this library was approximately 400million independent clones. After two days, the supernatant containingEEV was harvested and debris removed by low speed centrifugation. Theclarified supernatant was then spun at 13,000 rpm (28,000×g) for 1 hourto pellet the antibody-EEV. The antibody-EEV was resuspended in 1 mlEMEM with 10% FBS and stored at 4 degrees until ready for use. In orderto make the antigen virus for panning, BHK cells (2×10⁸ cells) wereinfected at a MOI=1.5 with virus expressing FZD4-ECD-A56R (SEQ ID NO: 6)in two cellSTACK cell culture chamber (Corning). After two days, thesupernatant containing EEV was harvested and debris removed by low speedcentrifugation. The clarified supernatant was then spun at 13,000 rpm(28,000×g) for 1 hour to pellet the antigen-EEV. The supernatant wasaspirated and the pellet resuspended in 1.0 mL of 1× PBS. The one mL ofthe FZD4-ECD-A56R EEV was transferred to a fresh tube and Psoralen(Trioxsalen, 4′-aminomethyl-, hydrochloride; Sigma) was added to 40μg/ml final concentration. The EEV and Psoralen were incubated at roomtemperature for 10 minutes before being irradiated in the STRATALINKER®UV Crosslinker (Stratagene) for 99,999 microjoules. The Psoralen/UVprocedure ensures that the antigen-EEV is inactivated and thereforeunable to form plaques or multiply in any downstream testing.

Tosylactivated MyOne DYNABEADS® (150 μL) were pulled down with a magnetand washed with 1 mL of PBS, two times. The beads were pulled down withthe magnet, the PBS removed and the 1 mL of Psoralen/UV inactivatedFZD4-ECD-A56R was added to the beads. The beads and antigen-EEV wereallowed to rotate at 37° C. for 18-20 hours. The beads were pelleted andthe supernatant was removed. The beads were blocked with 1 mL of 1× PBS,10% FBS and 0.5% BSA at 37° C. for 2 hours. The beads were pelleted andwashed with 1 mL 1× PBS before being resuspended in 150 μL of 1× PBS.

Fifty microliters of FZD4-ECD-A56R-coupled beads were added to 1 mL ofthe antibody-EEV library. The FZD4-ECD-A56R coupled beads and antibodyEEV were allowed to rotate at room temperature for 2 hours. Beads werepelleted using the magnet and unbound supernatant removed. The beadswere then washed five times with 1 mL of DMEM media supplemented with10% FBS and 1 mM HEPES (10% DMEM). All washes were pooled with theunbound supernatant (“Unbound”). The beads (“Bound”) were thenresuspended in 1 mL of 10% DMEM. “Unbound” and “Bound” were titered onBSC-1 cells and overlaid with growth medium containing methylcellulose.Plaques were allowed to form for two days and then the cells were fixedand stained with 0.1% Crystal Violet solution. The remaining bound virus(990 μl) was divided among 5 T175 flasks containing confluent BSC1 cellsand allowed to amplify in DMEM 2.5% containing 1 mg/ml G418 for 3 days.The cells were then harvested, and the virus released by three cycles offreeze/thaw, and the virus titered.

For the second round of selection (Rd2), the amplified Heavy chains fromround 1 were co-infected along with fresh L48 into one cellSTACK of BHK.Antibody EEV was harvested as described above. For each round ofpanning, fresh FZD-ECD-A56R antigen virus was produced, concentrated,inactivated and coupled to beads as described above. Fifty microlitersof FZD4-ECD-A56R coupled beads was added to 1 mL antibody-EEV Rd2. TheFZD4-ECD-A56R coupled beads and antibody EEV were allowed to rotate atroom temperature for 2 hours. Beads were pelleted using the magnet andunbound supernatant removed. The beads were then washed five times with1 mL of DMEM media supplemented with 10% FBS and 1 mM HEPES (10% DMEM).All washes were pooled with the unbound supernatant (“Unbound”). Thebeads (“Bound”) were then resuspended in 1 mL of 10% DMEM. “Unbound” and“Bound” were titered on BSC-1 cells and overlaid with growth mediumcontaining methylcellulose. Plaques were allowed to form for two daysand then the cells were fixed and stained with 0.1% Crystal Violetsolution. The remaining bound virus (990 ul) was divided among 5 T175flasks containing confluent BSC1 cells and allowed to amplify in DMEM2.5% containing 1 mg/ml G418 for 3 days. The cells were then harvested,and the virus released by three cycles of freeze/thaw, and the virustittered.

Three additional cycles of panning (Rd3, Rd4, and Rd5) were performed asdescribed above.

Rounds 3, 4 and 5 were tested for enrichment by infected A431 cells in 6well plate at a MOI=1 with each amplified VH round and L48. After anovernight infection the cells were harvested and split in half. One halfwas stained with 10 μg/ml FZD-His, followed by anti-His-Dyelight650 andanti-Fab-FITC. The other half was stained with 10 μg/ml CD100-His(negative control), followed by anti-His-Dyelight650 and anti-Fab-FITC.The data shown in FIG. 7 shows increasing enrichment per round ofselection. Antibodies from round 5 were sub-cloned into a mammalianexpression vector to be expressed as full length soluble IgG andtransfected (along with L48 in a mammalian expression vector). Theresulting antibodies present in the supernatant were tested by flowcytometry for binding to FZD4 transfected CHO cells and the absence ofbinding to CXCR4 transfected CHO cells. A number of antibodies thatbound specifically to FZD were identified.

Example 7: Dual Tag/Antigen EEV can be Coupled to Magnetic Beads

BHK cells were infected at two virions per cell where one virion wasHemagglutinin tag (HA)-A56R (SEQ ID NO: 17) and the second was FZD4-F13L(SEQ ID NO: 2) in order to yield EEV expressing both the HA tag and FZD4antigen on its surface or infected at one virion per cell with eachindividual virus. After two days, the supernatant containing EEV washarvested and debris removed by low speed centrifugation. Protein Gmagnetic beads (150 μL) were pulled down with a magnet and 1 mL ofPBS+30 μg of purified anti-FZD4 antibody (C6073) was added to the beads.The solution was incubated at room temperature with gentle rotation for25 minutes to allow the antibody to couple to the Protein G beads. Beadswere pulled down with the magnet, washed once with 1 mL of PBS andresuspended in 300 μL of DMEM+10% FBS. Anti-HA-tag magnetic beads(ThermoFisher, 150 μl) pulled down with a magnet and washed once with 1mL of PBS before resuspending in 150 μl of PBS.

HA-A56R (SEQ ID NO: 17) MGWSCIILFLVATATGAHS

TSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRKYKTENKVSingle Underline - Signal sequence (amino acids 1- 19)Bold - HA Tag (amino acids 20-29)Italics - Truncated A56R (amino acids 30-235)

Fifty μL of prepared anti-HA-tag beads or 100 μl of prepared anti-FZD4Protein G were added to 1 mL of each EEV supernatant and allowed torotate at room temperature for 60 minutes. Beads were pelleted using themagnet and unbound supernatant removed. The beads were then washed fivetimes with 1 mL of DMEM media supplemented with 10% FBS and 1 mM HEPES(10% DMEM). All washes were pooled with the unbound supernatant(“Unbound”). The beads (“Bound”) were then resuspended in 1 mL of 10%DMEM. “Unbound” and “Bound” were titered on BSC-1 cells and overlaidwith growth medium containing methylcellulose. Plaques were allowed toform for two days and then the cells were fixed and stained with 0.1%Crystal Violet solution. Plaques were counted to determine the number ofplaque forming units (pfu) in the “Unbound” and “Bound” from which the %of EEV bound to the beads could be calculated. Results are shown in FIG.8. EEV expressing both fusion proteins were pulled down by eitherantibody.

Example 8: Dual Tag/Antigen EEV can be Coupled to Magnetic Beads andUsed to Capture mAb EEV

BHK cells (2×10⁸ cells) were infected at two virions per cell where onevirion was HA-A56R (SEQ ID NO: 17) and the second was CXCR4-F13L (SEQ IDNO: 3) in order to yield EEV expressing both the HA tag and CXCR4antigen on its surface. After two days, the supernatant containing EEVwas harvested and debris removed by low speed centrifugation. Theclarified supernatant was then spun at 13,000 rpm (28,000×g) for 1 hourto pellet the tag/antigen-EEV. The supernatant was aspirated and thepellet resuspended in 1 mL of 1× PBS. Psoralen (Trioxsalen,4′-aminomethyl-, hydrochloride; Sigma) was added to 40 ug/ml finalconcentration. The EEV and Psoralen were incubated at room temperaturefor 10 minutes before being irradiated in the Stratalinker UVCrosslinker (Stratagene) for 99,999 microjoules twice. The Psoralen/UVprocedure ensures that the antigen-EEV is inactivated and thereforeunable to form plaques or multiply in any downstream testing.

Anti-CXCR4 EEV and anti-HER2 mAb EEV were produced by infecting BHKcells at two virions per cell where one virion was specific heavy chainand the second was specific light chain. After two days, supernatantscontaining the anti-CXCR4 EEV and the anti-HER2 EEV were harvested anddebris removed by low speed centrifugation.

Three hundred microliters of anti-HA magnetic beads were washed with 1mL of PBS and then resuspended in one milliliter of the Psoralen/UVinactivated HA/CXCR4 EEV. The beads and EEV were incubated at roomtemperature with gentle rotation for 90 minutes to allow the EEV tocouple with the anti-HA beads. Beads were pulled down with the magnet,washed once with 1 mL of PBS and resuspended in 300 μL of PBS.

One hundred μL of HA/CXCR4 EEV coupled to the anti-HA beads was added to1 mL of each mAb EEV supernatant and incubated at room temperature withgentle rotation for 1-1.5 hours. Beads were pelleted using the magnetand unbound supernatant removed. The beads were then washed five timeswith 1 mL of DMEM media supplemented with 10% FBS and 1 mM HEPES (10%DMEM). All washes were pooled with the unbound supernatant (“Unbound”).The beads (“Bound”) were then resuspended in 1 mL of 10% DMEM. “Unbound”and “Bound” were titered on BSC-1 cells and overlaid with growth mediumcontaining methylcellulose. Plaques were allowed to form for two daysand then the cells were fixed and stained with 0.1% Crystal Violetsolution. Plaques were counted to determine the number of plaque formingunits (pfu) in the “Unbound” and “Bound” from which the % of EEV boundto the beads could be calculated. Results are shown in FIG. 9.Anti-CXCR4 EEV were specifically captured by the beads coated with EEVco-expressing HA-A45R and CXCR4-F13L.

Example 9: Antigen-EEV can be Biotinylated for Coupling to MagneticBeads

BHK cells were infected at a MOI=1 with virus expressing FZD4-F13L,FZD4-ECD-A56R or CD20-F13L. After two days, the supernatant containingEEV was harvested and debris removed by low speed centrifugation. Theclarified supernatant was then spun at 13,000 rpm for 1 hour to pelletthe antigen-EEV. The supernatant was aspirated and the pelletresuspended in 1-2 mL of 1× PBS. To biotinylate the EEV, 2.5 μL ofBiotin-XX SSE stock solution in 1× PBS (FLUOREPORTER® Cell SurfaceBiotinylation Kit, Molecular Probes) was added to the 1 mL of each EEVin PBS and incubated on ice for 30 minutes. Fifty μL of 1M Tris, pH 8was added to quench each reaction.

To couple the Biotin-EEV to beads, 150 μL of Streptavidin DYNABEADS®were pelleted and washed once with 1 mL of 1× PBS. The beads wereresuspended in 150 μL and 50 μL was added to 1 mL of Eagle's MinimumEssential Medium (EMEM) media containing 10% FBS and 1 mM HEPES (10%EMEM). Fifty μL of Biotin-EEV was added to the beads and media andallowed to rotate at room temperature for 1 hour. Beads were pelletedusing the magnet and unbound supernatant removed. The beads were thenwashed five times with 1 mL of DMEM media supplemented with 10% FBS and1 mM HEPES (10% DMEM). All washes were pooled with the unboundsupernatant (“Unbound”). The beads (“Bound”) were then resuspended in 1mL of 10% DMEM. “Unbound” and “Bound” were titered on BSC-1 cells andoverlaid with growth medium containing methylcellulose. Plaques wereallowed to form for two days and then the cells were fixed and stainedwith 0.1% Crystal Violet solution. Plaques were counted to determine thenumber of plaque forming units (pfu) in the “Unbound” and “Bound” fromwhich the % of EEV bound to the beads could be calculated. Results areshown in FIG. 10.

Antigen-EEV was able to be biotinylated and coupled to magneticStreptavidin beads.

1. An isolated polynucleotide comprising: (a) a first nucleic acidfragment that encodes an integral membrane protein (IMP) or fragmentthereof, wherein the IMP or fragment thereof comprises at least oneextra-membrane region, at least one transmembrane domain and at leastone intra-membrane region, and wherein a portion of the first nucleicacid fragment encoding at least one intra-membrane region is situated atthe 5′ or 3′ end of the first nucleic acid fragment; and (b) a secondnucleic acid fragment that encodes a poxivirus EEV-specific protein orfunctional fragment thereof, wherein the second nucleic acid fragment isfused in frame to a portion of the first nucleic acid fragment thatencodes an intra-membrane region of the IMP; wherein a poxvirus infectedcell comprising the polynucleotide can express an IMP poxvirusEEV-specific protein fusion protein as part of the outer envelopemembrane of an extracellular enveloped virion (EEV).
 2. Thepolynucleotide of claim 1, wherein the IMP is a multi-pass membraneprotein comprising at least two transmembrane domains.
 3. Thepolynucleotide of claim 2, wherein the IMP has an odd number oftransmembrane domains, wherein the 5′ end of the first nucleic acidfragment encodes an extra-membrane region, wherein the 3′ end of thefirst nucleic acid fragment encodes an intra-membrane region, andwherein the 5′ end of the second polynucleotide is fused to the 3′ endof the first nucleic acid fragment.
 4. The polynucleotide of claim 3,wherein the IMP comprises a G-protein coupled receptor (GPCR).
 5. Thepolynucleotide of claim 4, wherein the IMP is the human frizzled-4protein (FZD4), or a fragment thereof.
 6. The polynucleotide of claim 4,wherein the IMP is the CXC chemokine receptor CXCR4, or a fragmentthereof.
 7. The polynucleotide of claim 2, wherein the IMP has an evennumber of transmembrane domains, and wherein both the 5′ and 3′ ends ofthe first nucleic acid fragment encode intra-membrane regions, andwherein the second nucleic acid fragment is fused to 3′ end of the firstnucleic acid fragment.
 8. The polynucleotide of claim 7, wherein the IMPis human CD20 protein, or a fragment thereof.
 9. The polynucleotide ofclaim 1, which is operably associated with a poxvirus promoter.
 10. TheIMP poxvirus EEV-specific protein fusion protein encoded by thepolynucleotide of claim
 1. 11. A poxvirus genome comprising thepolynucleotide of claim
 1. 12. (canceled)
 13. (canceled)
 14. (canceled)15. (canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)24. A recombinant poxvirus EEV comprising a heterologous IMP or fragmentthereof fused to a poxvirus EEV-specific protein or membrane-associatedfragment thereof, wherein the fusion protein is situated in the EEVouter envelope membrane, wherein the IMP or fragment thereof displays onthe surface of the EEV in its native conformation.
 25. (canceled)
 26. Amethod to select antibodies that bind to a multi-pass membrane proteincomprising: (a) attaching the recombinant EEV of claim 24 to a solidsupport; (b) providing an antibody display library, wherein the librarycomprises display packages displaying a plurality of antigen bindingdomains; (c) contacting the display library with the EEV such thatdisplay packages displaying antigen binding domains that specificallybinds to the IMP expressed on the EEV can bind thereto; (d) removingunbound display packages; and (e) recovering display packages thatdisplay an antigen binding domain specific for the IMP expressed on theEEV.
 27. The method of claim 26, wherein the EEV are attached to thesolid surface via reaction with tosyl groups attached to the surface.28. The method of claim 26, wherein the EEV are biotinylated andattached to a streptavidin coated solid surface.